Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer

ABSTRACT

The present invention relates to methods and apparatuses for analyzing molecules, particularly polymers, and molecular complexes with extended or rod-like conformations. In particular, the methods and apparatuses are used to identify repetitive information in molecules or molecular ensembles, which is interpreted using an autocorrelation function in order to determine structural information about the molecules. The methods and apparatuses of the invention are used for, inter alia, determining the sequence of a nucleic acid, determining the degree of identity of two polymers, determining the spatial separation of specific sites within a polymer, determining the length of a polymer, and determining the velocity with which a molecule penetrates a biological membrane.

This application claims the benifit of to U.S. Provisional ApplicationSer. No. 60/096,543, filed Aug. 13, 1998, which is incorporated byreference herein in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to methods and apparatuses for analyzingmolecules, particularly polymers, and molecular complexes with extendedor rod-like conformations. In particular, the methods and apparatusesare used to identify repetitive information, e.g., sequence information,in molecules or molecular ensembles, which is subsequently used todetermine structural information about the molecules. The methods arebased on the use of an autocorrelation function to identify commoninformation in multiple molecules having at least one overlappingrepetitive sequence.

2. BACKGROUND OF THE INVENTION

Macromolecules are involved in diverse and essential functions in livingsystems. The ability to decipher the functions, dynamics, andinteractions of macromolecules is dependent upon an understanding oftheir chemical and three-dimensional structures. These threeaspects—chemical and three-dimensional structures and dynamics—areinterrelated. For example, the chemical composition of a protein, andmore particularly the linear arrangement of amino acids, explicitlydetermines the three-dimensional structure into which the polypeptidechain folds after biosynthesis (Kim & Baldwin (1990) Ann. Rev. Biochem.59: 631-660), which in turn determines the interactions that the proteinwill have with other macromolecules, and the relative mobilities ofdomains that allow the protein to function properly.

Biological macromolecules are either polymers or complexes of polymers.Different types of macromolecules are composed of different types ofmonomers, i.e., twenty amino acids in the case of proteins and fourmajor nucleobases in the case of nucleic acids. A wealth of informationcan be obtained from a determination of the linear, or primary, sequenceof the monomers in a polymer chain. For example, by determining theprimary sequence of a nucleic acid, it is possible to determine theprimary sequences of proteins encoded by the nucleic acid, to generateexpression maps for the determination of mRNA expression patterns, todetermine protein expression patterns, and to understand how mutationsin genes correspond to a disease state. Furthermore, the characteristicpattern of distribution of specific nucleobase sequences along aparticular DNA polymer can be used to unequivocally identify the DNA, asin forensic analysis.

In general, DNA identification and sequencing has been performed usingmethods, such as those described by Maxam and Gilbert (Maxam & Gilbert(1977) Proc. Natl. Acad. Sci. U.S.A. 74: 560-564) and by Sanger (Sangeret al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467) thatdetermine the exact sequence of relatively short pieces of DNA. Thereare also techniques that arrange these short DNA fragments of knownsequence in the proper order to obtain a longer sequence, such as thosedescribed by Evans (U.S. Pat. No. 5,219,726). Other methods of nucleicacid detection and sequencing have been developed, however, these toohave limitations in the number of nucleotides they can read, in theirabilities to resolve the identities of adjacent nucleotides, and in thepracticality of their implementation.

Several methods for rapid sequencing of nucleic acids have beendeveloped that use exonucleases to cleave individual bases from thenucleic acid polymer, which are subsequently identified in order togenerate the sequence of the nucleic acid. U.S. Pat. No. 4,962,037discloses a method wherein the nucleic acid fragment is suspended in aflowing stream while an exonuclease sequentially cleaves individualbases from the end of the fragment. The flowing stream delivers thecleaved bases in an ordered fashion to a detector for subsequentidentification. A similar approach with some modifications is disclosedin U.S. Pat. No. 5,674,743. In this method, the DNA strand to besequenced is processed with an exonuclease to cleave bases from thestrand, and each cleaved base is then transported away from the strandand is incorporated into a fluorescence-enchancing matrix. In aparticular embodiment, the intrinsic fluorescence of the nucleotide isinduced and is used to identify it. Using a processive exonuclease, itis theoretically possible to sequence 10,000 bases or more at a rate of10 bases per second. However, exonuclease sequencing has encounteredmany problems. If extrinsic labels are used to identify each base, allfour bases must be tagged with, e.g., different fluorophores, which issterically difficult; in addition, introduction of fluorophores mayinterfere with the enzymatic activity of the exonuclease. Furthermore,difficult optical trapping is needed to suspend DNA molecules in aflowing stream. Lastly, single molecules of fluorophore need to bedetected with high efficiency, and only 95% efficiency has beenachieved.

Methods of nucleic acid sequencing by hybridization with a specific setof oligonucleotide probes are also known in the art (Strezoska et al.(1991) Proc. Natl. Acad. Sci. U.S.A. 88: 10089-10093; Bains (1992)BioTechnology 10: 757-758). Although this approach is very costly to setup, sequencing by these methods is ultimately low-cost ($0.03-0.08 perbase). Another advantage is the potential integration of the techniquewith microelectronics using special microchips for sequencing of nucleicacids fragments and even analysis of entire genomes (Service (1998)Science 282: 396-399 & 399-401). Traditional sequencing by hybridizationtechniques have the limitation of imperfect hybridization, especiallyunder conditions in which hybridization is not favored, e.g., low-salt,or upon formation of secondary structure in the target nucleic acid,which interferes with binding to the probes. Imperfect hybridizationleads to difficulties in generating adequate sequence because the errorin hybridization is amplified many times.

U.S. Pat. No. 5,846,727 discloses a microsystem for rapid DNA sequencingin which a DNA template is amplified using the polymerase chain reaction(“PCR”) and the PCR products are labeled and immobilized on a capillarytube wall. Then, Sanger extension products of the amplified DNA areprepared, labeled, and electrophoretically separated in a capillarychannel. Near-infrared, laser-induced fluorescence of theoligonucleotides is detected. The same fluorophore is used to label allbases; however, different bases can be distinguished by difference ofthe fluorescence lifetimes induced by different bases upon the labeling.The substrate used is selected for compatibility with both the solutionsand the conditions to be used in analysis, including but not limited toextremes of salt concentrations, acid or base concentration,temperature, electric fields, and transparence to wavelengths used foroptical excitation or emission. The substrate material may include thoseassociated with the semiconductor industry, such as fused silica,quartz, silicon, or gallium arsenide, or inert polymers such aspolymethylmetacrylate, polydimethylsiloxane, polytetrafluoroethylene,polycarbonate, or polyvinylchloride. Because of its transmissiveproperties across a wide range of wavelengths, quartz is a preferredembodiment.

The use of quartz as a substrate with an aqueous solution means that thesurface in contact with the solution has a positive charge. When workingwith charged molecules, especially under electrophoresis, it isdesirable to have a neutral surface. In one embodiment, a coating isapplied to the surface to eliminate the interactions which lead to thecharge. The coating may be obtained commercially (capillary coatings bySupelco, Bellafonte Pa.), or it can be applied by the use of a silanewith a functional group on one end. The silane end will bond effectivelyirreversibly with the glass, and the functional group can react furtherto make the desired coating. For DNA, a silane with polyethyleneoxideeffectively prevents interaction between the polymer and the wallswithout further reaction, and a silane with an acrylamide group canparticipate in a polymerization reaction to create a polyacrylamidecoating which not only does not interact with DNA, but also inhibitselectro-osmotic flow during electrophoresis.

The microchannels may be constructed on the substrate by any number oftechniques, many derived from the semiconductor industry, depending onthe substrated selected. These techniques include, but are not limitedto, photolithography, reactive ion etching, wet chemical etching,electron beam writing, laser or air ablation, LIGA, and injectionmolding. A variety of these techniques applied to polymer-handling chipshave been discussed in the literature including by Harrison et al.(Analytical Chemistry 1992 (64) 1926-1932), Seiler et al. (AnalyticalChemistry 1993 (65) 1481-1488), Woolley et al. (Proceedings of theNational Academy of Sciences November 1994 (91) 11348-11352), andJacobsen et al. (Analytical Chemistry 1995 (67) 2059-2063). Thedisclosed microsystem offers several advantages like the need of onlysub-microliter volumes of expensive reagents, the ability to automatethe procedure and perform several analyses simultaneously, and the useof a “highly efficient base-calling scheme using a single lane,single-dye format”. Despite these advantages, typical read lengths ofthis method are still only on the order of 400-500 bases.

There are several other methods (U.S. Pat. No. 4,962,037 and U.S. Pat.No. 5,674,743, see below) that can be used to sequence long DNAmolecules. However, the maximal length of a single DNA fragment that canbe sequenced by existing techniques is still less than 2,000 bases(Mullikin & McMurray (1999) Science 283: 1867-1868; Sinclair (1999) TheScientist 15 (9): 18-20).

Methods have also been developed for quantitative detection ofmacromolecules in a sample. Recent developments in experimentaltechniques and available hardware have increased dramatically thesensitivity of detection so that optical measurements can be made ofeven single molecules in a sample. Such measurements can be done inaqueous solution, at room temperature (Weiss (1999) Science 283:1676-1683), and in very small volumes to reduce background scattering.

Fluorescence correlation spectroscopy (“FCS”) uses an autocorrelationfunction to process fluctuations in fluorescence emission from arestricted volume (Elson & Magde (1974) Biopolymers 13:1-27). Thisapproach is essentially based on the assumptions that: (a) one or zerofluorescent molecules can be within an illuminated volume; and (b) thefluorescence emitted by the fluorescent molecule in the illuminatedvolume noticeably exceeds background. The detected fluorescent bursts,whose lengths are related to the time a molecule spends within theilluminated volume, can be used to identify and count molecules, as wellas to determine diffusion coefficients (U.S. Pat. No. 4,979,824, WO Pat.No. 94/16313, the latter patent uses FCS).

Eigen & Rigler [(1994) Proc. Natl. Acad. Sci. U.S.A. 91: 5740-5747],describe the use of FCS for parallel screening of large amounts ofgenetic material for a particular sequence pattern. In particular, theinteraction of a fluorescent ligand, e.g., a labeled oligonucleolide,with a larger target DNA can be measured by the correlation functiondescribing the diffusion of the free and bound ligand. Anoligonucleotide hybridized with a large DNA fragment would diffuse moreslowly than free oligonucleotide, and therefore, the bound form of thefluorescent oligonucleotide exhibits longer photon bursts. Amodification of this technique uses the cross correlation of signalsobtained from different oligonucleotides labeled with differentfluorophores to detect the presence of different oligonucleotidesequences within a DNA target sample (Schwille et al. (1997) Biophys. J.72: 1878-1886).

PCT Publication No. WO 98/10097 discloses a method and apparatus fordetection of single molecules emitting two-color fluorescence anddetermination of molecular weight and concentration of the molecules.The method involves the labeling of individual molecules with at leasttwo fluorescent probes. The velocity is determined by measuring the timerequired for the molecules to travel a fixed distance between two laserbeams. Comparison of the molecule's velocity with that of standardspecies permits determination of the molecular weight of the molecule,which may be present in a concentration as small as one femtomolar. Theaccuracy of the technique is limited by the time the molecule underscrutiny spends traveling through the spot of the focused laser beam.The diameter of the laser beam is diffraction limited and exceeds 0.4 μmfor visible light.

Castro and Shera [(1995) Anal. Chem. 67: 3181-3186] describe the use ofsingle molecule electrophoresis (SME) for the detection andidentification of single molecules in solution. The technique involvesthe determination of electrophoretic velocities by measuring the timerequired for individual molecules labeled with a single fluorophore totravel a fixed distance between two laser beams. This technique has beenapplied to DNA, to fluorescent proteins and to simple organicfluorophores. An advantage of SME over conventional zone electrophoresisis that SME is a continuous flow system that permits real-time analysis,which is important when sample concentration and/or composition changeswith time. The disclosed system has disadvantages when applied to thedetection of a specific DNA sequence within a large genomic background.If a single fluorescent probe complementary to the sequence of interestis used, it can bind non-specifically to other sequences in the genomicDNA, which results in detection of a false positive. Moreover, anunbound probe also produces a detectable signal that could bemisinterpreted as the presence of the target sequence.

U.S. Pat. No. 5,807,677 discloses a method and device for directidentification of a specific target nucleic acid sequence having a lowcopy number in a test solution. This method involves the preparation ofa reference solution of a mixture of different short oligonucleotides.Each oligonucleotide includes a sequence complementary to a section ofthe target sequence and is labeled with one or more fluorescent dyemolecules. The reference solution is incubated with the test solutionunder conditions favorable to hybridization of the shortoligonucleotides with the nucleic acid target. The target sequence isidentified in the solution by detection of the nucleic acid strands towhich one or more of the labeled oligonucleotides are hybridized. Toamplify the fluorescence signal, a “cocktail” of differentoligonucleotides are used which are capable of hybridizing withsequences adjacent to but not overlapping with the target sequence. Thedisadvantage of this method is that, in order to design probes of theproper sequence, the exact sequence of the target nucleic acid andsurrounding sequences must be known.

PCT Publication No. WO 96/06189 describes a method for quantitativedetection of oligonucleotides using capillary electrophoresis.Typically, capillary electrophoresis employs fused silica capillarytubes whose inner diameters are between about 10-200 microns, and whichcan range in length between about 5-100 cm or more. As the innerdiameter of such a capillary is small, electric fields 10 to 100 timesstronger than those applicable in conventional electrophoretic systemscan be applied because of reduced Joule heating. This permits very highspeeds and superior resolution. In the methods described in PCTPublication No. WO 96/06189, a fluorescently labeled peptide nucleicacid ranging in size from 5-50 monomers is hybridized to a DNA sampleand capillary electrophoresis through a polyacrylamide gel is performedunder denaturing conditions (7 M urea) where the PNA/DNA complex isstable. This method suffers from limited detection sensitivity andcannot be used to detect single copy genes in large genomes.

The existing methods for sequencing polymers and for detecting thepresence of small amounts of specific polymers in a sample each havedrawbacks. The major drawbacks of sequencing techniques are that theyare slow, labor intensive, and have fairly short read lengths (under2,000 bases for nucleic acid sequencing) and limited accuracy. Themethods for detecting molecules in a sample have the drawbacks of lackof sensitivity, frequent occurrence of false positive results, and, insome cases, a requirement that the sequence of the molecule to bedetected must already be known. Clearly, there is a need for faster,simpler, more reliable and more universally applicable methods ofsequencing and of detecting copies of sequences in a sample in order toelucidate complex genetic function and diagnose diseases and geneticdysfunctions more rapidly and accurately.

Citation of a reference herein shall not be construed as indicating thatsuch reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION

In a first embodiment, the present invention relates to a method foranalyzing an extended object comprising: (a) moving with respect to atleast one station a plurality of similar extended objects that are eachsimilarly labeled with at least two unit-specific markers to generate aplurality of object-dependent impulses as the labeled extended objectspass the station; (b) measuring the generated plurality ofobject-dependent impulses as a function of one or more systemparameters; and (c) calculating an autocorrelation function of saidobject-dependent impulses, to analyze the extended object.

In a second embodiment, the present invention relates to a method foranalyzing an extended object comprising calculating an autocorrelationfunction of object-dependent impulses.

In a third embodiment, the present invention relates to an article ofmanufacture comprising a lattice of spherical beads having a pluralityof fixed stations with at least one fluorophore positioned at each fixedstation.

In a fourth embodiment, the present invention relates to a system foranalyzing an extended object labeled with at least two unit-specificmarkers comprising: a central processing unit; an input device forinputting a plurality of object-dependent impulses of an extendedobject; an output device; a memory; at least one bus connecting thecentral processing unit, the memory, the input device, and the outputdevice; the memory storing a calculating module configured to calculatean autocorrelation function for said plurality of object-dependentimpulses of said extended object input using said input device.

In a fifth embodiment, the present invention relates to a computerprogram product for use in conjunction with a computer, the computerprogram product comprising a computer readable storage medium and acomputer program mechanism embedded therein, the computer programmechanism comprising a calculating module configured to calculate anautocorrelation function of a plurality of object-dependent impulses.

The methods, articles of manufacture, computer system, and computerprogram products of the invention are useful for analyzing polymers,particularly DNA.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Outline of the method of correlated FRET. D and A are donorand acceptor fluorophores, respectively; L and V are length and velocityof movement of the labeled DNA fragment; 1_(ex), 1_(emD), and 1_(emA)are the wavelengths of the donor excitation, the donor emission, and theacceptor emission, respectively; T₀ and T₀+ΔT are the times when thefirst and the last acceptor fluorophores pass the D-center,respectively.

FIG. 2. A schematic diagram illustrating the restricted movement of anextended flexible molecule within a network.

FIGS. 3A-3B. A schematic diagram depicting the hybridization sequencingwith distance information: dsDNA is a double stranded DNA fragment to besequenced; Greek letters denote probing oligonucleotides withfluorescence labels. Panel 3A: pairs of labeled probe oligomers are usedin every run and L_(ij) is the distance between the probes. Panel 3B: anend of the target DNA is labeled, a single labeled probe oligomer isused in every run and L_(ij) is the distance between the labeled end ofthe DNA and the probe.

FIG. 4. An example of an optical setup for measurement of correlatedFRET. L=laser; A=attenuator; LF=line filter; E=beam expander;DM=dichroic mirror; SL=sample lens; S=sample; DL=detector lens;BF=bandpass filter; P=pinhole; PD=photodetector; MA=multichannelanalyzer; and C=computer.

FIGS. 5A-5C. A schematic diagram of structures for performing themethods of analyzing extended objects using an autocorrelation function.Panel 5A: a gel matrix having D-centers bound within the matrix; Panel5B: a lattice of spheres, wherein the D-centers are fluorescentlylabeled spheres; and Panel 5C: a nanochannel plate wherein the D-centeris a thin film of donor fluorophores.

FIG. 6. A schematic diagram depicting the geometric limitations of theresolution of gel-bound D-centers.

FIG. 7. A schematic diagram depicting the geometric limitations of theresolution of fluorescent beads as D-centers.

FIG. 8. shows examples of various structures that fall within the scopeof the invention.

FIG. 9. shows a configuration for consistent unraveling, delivery, andstretching of DNA of varying sizes.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Introduction

The present invention relates to methods of analyzing polymers as wellas molecules and molecular complexes with extended or rod-likeconformations. All objects to be analyzed are hereinafter referred to as“extended objects.” In particular, the method is intended to identifyrepetitive information in the extended objects or in ensembles ofextended objects. The extended objects are similar, or preferably, thesame, and comprise a similar, or preferably, an identical pattern oflabeled units. The labeled extended objects are moved past at least onestation, at which labeled units of the extended objects interact withthe station to produce an object-dependent impulse. Preferably, labeledextended objects are moved past a plurality of stations. Because theextended objects are similar, or preferably identical, and comprise asimilar, or preferably, identical pattern of labeled units, acharacteristic signature of interactions is repeated as each extendedobject moves past a station or a plurality of stations. This repetitiveinformation is extracted from the overall raw data by means of anautocorrelation function and is then used to determine structuralinformation about the extended objects. As used in this application,“moves past” refers to embodiments in which the station is stationaryand the extended object is in motion, the station is in motion and theextended object is stationary, and the station and extended object areboth in motion; all such embodiments are within the scope of theinvention.

In the preferred embodiment, the extended object to be analyzed is apolymer. A polymer, as used herein, is a compound having a linearbackbone of individual units linked together by covalent bonds.Preferably, the backbone is unbranched. The term “backbone” is given itsusual meaning in the field of polymer chemistry. The polymers may beheterogeneous in backbone composition, thereby containing any possiblecombination of individual monomer units linked together, e.g.,peptide-nucleic acids (PNA), having a polypeptide-like backbone, basedon the monomer 2-aminoethyleneglycin carrying any of the fournucleobases: A, T, G, or C. In a preferred embodiment, the polymers arehomogeneous in backbone composition and are, e.g., nucleic acids orpolypeptides. A nucleic acid as used herein is a biopolymer comprised ofnucleotides, such as deoxyribose nucleic acid (DNA) or ribose nucleicacid (RNA). A protein or polypeptide as used herein is a biopolymercomprised of amino acids. In the most preferred embodiment, the extendedobject is a double-stranded DNA molecule with a rigid structure.

As used herein with respect to individual units of a polymer, “linked”or “linkage” means two units are joined to each other by anyphysicochemical means. Any linkage known to those of ordinary skill inthe art, covalent or non-covalent, is embraced. Natural linkages, e.g.,amide, ester, and thioester linkages, which are those ordinarily foundin nature to connect the individual units of a particular polymer, aremost common. However, the individual units of a polymer analyzed by themethods of the invention may be joined by synthetic or modifiedlinkages.

A polymer is made up of a plurality of individual units, which arebuilding blocks or monomers that are linked either directly orindirectly to other building blocks or monomers to form the polymer. Thepolymer preferably comprises at least two chemically distinct linkedmonomers. The at least two chemically distinct linked monomers mayproduce or be labeled to produce different signals. Different types ofpolymers are composed of different monomers. For example, DNA is abiopolymer comprising a deoxyribose phosphate backbone to which areattached purines and pyrimidines such as adenine, cytosine, guanine,thymine, 5-methylcytosine, 2-aminopurine, hypoxantine, and othernaturally and non-naturally occurring nucleobases, substituted andunsubstituted aromatic moieties. RNA is a biopolymer comprising a ribosephosphate backbone to which are attached purines and pyrimidines such asthose described for DNA but wherein uracil is substituted for thymidine.Deoxyribonucleotides may be joined to one another via an ester linkagethrough the 5′ or 3′ hydroxyl groups to form the DNA polymer.Ribonucleotides may be joined to one another via an ester linkagesthrough the 5′, 3′ or 2′ hydroxyl groups. Alternatively, DNA or RNAunits having a 5′, 3′ or 2′ amino group may be joined via an amidelinkage to other units of the polymer.

The polymers may be naturally-occurring or non-naturally occurringpolymers. Polymers can be isolated, e.g., from natural sources usingbiochemical purification techniques. Alternatively, polymers may besynthesized, e.g., enzymatically by in vitro amplification using thepolymerase chain reaction (PCR), by chemical synthesis, or byrecombinant techniques.

The methods of the invention are performed by detecting signals referredto as object-dependent impulses. An “object-dependent impulse,” as usedherein, is a detectable physical quantity which transmits or conveysinformation about the structural characteristics of at least oneunit-specific marker of an extended object. A unit-specific marker, asused herein, can either be a measurable intrinsic property of aparticular type of individual unit of the extended object, e.g., thedistinct absorption maxima of the naturally occurring nucleobases of DNA(the polymer is intrinsically labeled), or a compound having ameasurable property that is specifically associated with one or moreindividual units of a polymer (the polymer is extrinsically labeled). Aunit-specific marker of an extrinsically labeled polymer may be aparticular fluorescent dye with which all nucleobases of a particulartype, e.g., all thymine nucleobases, in a DNA strand are labeled.Alternatively, a unit-specific marker of an extrinsically labeledpolymer may be a fluorescently labeled oligonucleotide of defined lengthand sequence that hybridizes to and therefore “marks” the complementarysequence present in a target DNA. Unit-specific markers may furtherinclude, but are not limited to, sequence specific major or minor groovebinders and intercalators, sequence-specific DNA or peptide bindingproteins, sequence specific PNAs, etc. The detectable physical quantitymay be in any form which is capable of being measured. For instance, thedetectable physical quantity may be electromagnetic radiation, chemicalconductance, radioactivity, etc. The object-dependent impulse may arisefrom energy transfer, directed excitation, quenching, changes inconductance (resistance), or any other physical changes. Although anobject-dependent impulse is specific for a particular unit-specificmarker, an object comprising more than one unit-specific marker willhave more than one identical object-dependent impulse. Additionally,chemically distinct unit-specific markers will give rise to differentobject-dependent impulses.

The method used for detecting the object-dependent impulse depends onthe type of physical quantity generated. For instance, if the physicalquantity is electromagnetic radiation, then the object-dependent impulseis detected optically. An “optically detectable” object-dependentimpulse as used herein is a light-based electromagnetic radiation signalthat can be detected by light detecting imaging systems. When thephysical quantity is chemical conductance, then the object-dependentimpulse is chemically detected. A “chemically detected” object-dependentimpulse is a signal in the form of a change in chemical concentration orcharge, such as ion conductance, which can be detected by standard meansfor measuring chemical potential or conductance. If the physicalquantity is an electrical signal then the object-dependent impulse is inthe form of a change in resistance or capacitance.

An object-dependent impulse arises from a detectable physical change inthe unit-specific marker of the extended object, in the station, or inthe environment surrounding the station. As used herein, a “detectablephysical change” in the unit-specific marker or the station is any typeof change that occurs in the unit-specific marker or the station as aresult of exposing the unit-specific marker to the station. When theunit-specific marker is exposed to the station, a detectable signal iscreated. The station may be an interaction station or a signalgeneration station. The type of change that occurs in the station or inthe unit-specific marker to produce the detectable signal depends on thetype of station and unit-specific marker used. Several examples ofcombinations of station and unit-specific markers that producedetectable signals are discussed herein below. Those of skill in the artwill be able to derive other combinations of stations and unit-specificmarkers that fall within the scope of the invention.

A “signal generation station” as used herein is a station that is anarea where the unit-specific marker interacts with the station or theenvironment around the station to generate an object-dependent impulse.In one embodiment of the invention, the object-dependent impulse resultsfrom contact in a defined area with an agent selected from the groupconsisting of electromagnetic radiation, a quenching source, and afluorescence excitation source which can interact with the unit-specificmarker to produce a detectable signal. In another embodiment, theobject-dependent impulse results from contact in a defined area with achemical environment that is capable of undergoing specific changes inconductance in response to an interaction with a chemically distinctstructure. As the chemically distinct structure interacts with thechemical environment, a unique change in conductance occurs. Thestructure-specific change may be temporal, e.g., the length of timerequired for the conductance to change, or it may be physical, e.g., themagnitude of an intensity change. In yet another embodiment, theobject-dependent impulse results from changes in capacitance orresistance caused by the movement of the unit-specific marker betweenmicroelectrodes or nanoelectrodes positioned adjacent to the object. Forexample, the signal generation station may include microelectrodes ornanoelectrodes positioned on opposite sides of the object such that aparticular change in conductance (resistance) that occurs as a result ofthe movement of a unit-specific marker past the electrodes will bespecific for the particular unit-specific marker.

Although the scope of the invention encompasses the detection of anyuseful physical changes, it is preferable to detect particular types ofphysical changes. For example, it is most preferable to carry out themethods of the present invention at room temperature and with theextended object dissolved in a solvent. The scale of thermalfluctuations at 300 K (room temperature) is ΔE_(T)=1/40 eV; therefore,the energy of an object-dependent impulse must considerably exceed thisvalue in order to be resolved from the thermal noise. Therefore, opticaldetection is most preferable, as the energy of a photon with awavelength in the visible light range is approximately 1 eV. Indeed,reliable detection of single photons by various means is well known inthe art. In the case of changes in conductivity, the scale of the effectis determined by ΔE_(C)=q²/2C, where q is the charge transferred due tothe object-dependent impulse and C is the capacitance of the electrodesused for the measurement. Using electron beam lithography, theelectrodes can be manufactured as small as 30×30 nm², which correspondsto a capacitance of approximately 3×10⁻¹⁷ F, and the thermal fluctuationamplitude at room temperature is equal to the transfer of 10 electrons.This limits the minimal change in ion concentration that must bemeasured by the electrodes in order to produce a signal that isdetectable above background. Moreover, biomolecules are studied inbuffered solutions, and the measurement of conductivity in a smallvolume of salt solution is additionally hindered by electrokineticeffects. In contrast, the major requirement for optical detection isthat the surrounding media be optically transparent within the spectralrange of detection. Therefore, a high energy per unit (photon) and therelative insensitivity to the properties of the surrounding media makeoptical changes the most preferred physical change to detect anobject-dependent impulse.

Various methods and products are available for analyzing extendedobjects, as described in PCT Publication No. WO 98/35012, which isincorporated herein by reference in its entirety.

Various optical effects that can be measured differ in their potentialsensitivity and resolution, i.e., the minimum distance between twoobjects wherein the objects are distinguishable. A low resolutioncorresponds to a larger distance between distinguishable objects; a highresolution corresponds to a smaller distance between distinguishableobjects. The resolution of a particular technique is determined by thecharacteristic distance through which the station may sense theparticular unit-specific marker of the extended object. A shortercharacteristic distance makes for better resolution. The lowestresolution techniques include monitoring of light transmission anddirected excitation. In the both cases, a source of light is employed,and it is the size of the light source that limits resolution. Theminimum size of a light source used in near-field optics exceeds 10 nm,and the effective size of the source increases exponentially with thedistance between the source and the object to be illuminated. Therefore,a resolution of 50-100 nm or more is known in the art (Tan & Kopelman(1996) Chem. Anal. Ser. 137: 407-475.). The resolution of quenchingtechniques is dictated by the size of the fluorophore that is in contactwith the quencher. However, the signal change is detected on a “brightbackground,” which decreases its sensitivity. Also, the quenching of afluorophore by other components in the solution, its bleaching, or itstransition to a long-lived triplet state is indistinguishable from itsquenching. Finally, it is difficult to design an experimental set upwherein all unit-specific markers of the extended object have directcontact with the same quenching group, which limits the utility of astation comprising a contact quencher.

5.2 Correlated Fluorescence Resonance Energy Transfer

In a preferred embodiment, the object-dependent impulses produced by theinteraction between the labeled units of the extended object and thestation arise as a result of fluorescence resonance energy transfer(“FRET”). FRET occurs when two fluorophores are in close proximity andwhen the emission spectrum of one fluorophore, the donor D, overlapswith the excitation spectrum of the other fluorphore, the acceptor A,and when D is in an excited state. The rate of energy transfer k_(T) isgiven by:

 k _(T)=1/τ_(D)(Z _(F) /Z)⁶  (1)

where τ_(D) is the fluorescence lifetime of D, Z is the distance betweenD and A, and Z_(F) is the Förster radius. When Z=Z_(F), the rates ofemission and energy transfer are equal, and 50% of excited donors aredeactivated by energy transfer. The value of Z_(F) is calculated fromspectroscopic parameters of D and A and takes into account theirrelative orientation. The utility of FRET is derived from the strongdependence of the efficiency of energy transfer on the sixth power ofthe distance between D and A. The most efficient energy transfer occurswhen the distance between D and A is close to Z_(F), which is betweentwo and seven nanometers for most organic fluorescent dye pairs (Wu &Brand (1994) Anal. Biochem. 218:1-13). Because of this strong distancedependence, FRET is often referred to as a “spectroscopic ruler” that isable to measure distances in the nanometer range (Stryer (1978) Ann.Rev. Biochem. 47:819-846).

In the methods of the present invention, FRET data is analyzed using anautocorrelation function. In one embodiment, the extended object islabeled with fluorescent donors D and the station is labeled withfluorescent acceptors A. In another embodiment, the extended object islabeled with fluorescent acceptors A and the station is labeled withfluorescent donors D. The movement of the extended object is such thateach of the unit-specific markers (D or A) on the object is moved withinthe proper distance of the A or D at the station for FRET to occur. TheDs are illuminated with light at the wavelength of excitation of thedonor. Emitted light is monitored within the wavelength band of emissionof the As and includes background emission and photons emitted by the Asdue to FRET from the Ds.

The movement of the extended objects to be analyzed is dominant in thedirection of the long axis of the extended object, while fluctuation inthe plane perpendicular to the long axis is much smaller (FIG. 1A). Foran extended object having multiple unit-specific markers, FRET from theunit-specific marker at the leading end of the object (FIG. 1B) isalways followed by FRET from the unit-specific marker at the trailingend of the object (FIG. 1C) as each one passes a given station. For anextended object having one unit-specific marker FRET at the firststation along the object's path is always followed by FRET at the secondstation along the object's path if: (a) one pair of stations is presentand all objects are moving along the path of this pair of stations; or(b) there are many pairs of stations along the path of the extendedobjects and the inter-station distance is the same in all pairs ofstations. Therefore, bursts of photons from these FRET events arecorrelated. This correlation allows the FRET events to be distinguishedfrom background emission by means of an autocorrelation function. In apreferred embodiment, the autocorrelation function is defined by formula2: $\begin{matrix}{{G(\tau)} = {{1/T}{\int_{0}^{T}{{I(t)}{I\left( {t + \tau} \right)}\quad {t}}}}} & (2)\end{matrix}$

wherein G(τ) is the autocorrelation function of the measuredembodiment-dependent time dependence of emission intensity I(t), and Tis the total time of measurement of I(t). In its simplest form, when thephoton counting mode is used, I(t) has a value of one at the time when aphoton is detected and is zero at other times. For background photonsmeasured at time t, there may or may not be another photon at time t+τ.Therefore, the product I(t)I(t+τ) may be either zero or one. At lowerbackground levels, the proportion of zero products is greater. Incontrast, for correlated events, a photon emitted at time t is alwaysaccompanied by a photon emitted at time t+τ₀, where τ₀=L/V, the intervalof correlation wherein L is the distance between labels and V is thevelocity of the extended object. The autocorrelation function G(τ)exhibits a maximum at τ=τ₀, the interval of correlation, and enableseither L or V to be calculated, depending on which of these parametersis known. Although the autocorrelation function is discussed herein inrelation to FRET analysis, one with skill in the art will recognize thatthis form of data analysis is applicable to other embodiments of theinvention and that the autocorrelation function can be a function of oneor more system parameters other than time.

One of skill in the art will recognize that although formula 2 iswritten for a continuous emission intensity I, the autocorrelationfunction may also be calculated for a discrete emission intensity:$\begin{matrix}{G_{j} = {\left( {1/N} \right){\sum\limits_{i = 0}^{N}{I_{i}I_{i + j}}}}} & (3)\end{matrix}$

where I_(i) corresponds to the moment t_(i), I_(i+j) corresponds to themoment t_(i)+jΔt, G_(j) is the autocorrelation function at time τ=jΔt,and N is the total number of data values in the I(t) sequence.

The application of an autocorrelation function for data analysis removesvolume and concentration restrictions from the experimental design. Thevolume of the experiment is only limited by the background emissiongenerated using a particular experimental set up. The resolution of themethod is determined by the scale on which the monitored interactionbetween station and object occurs, e.g., nm, μm, mm, cm, etc.

In a preferred embodiment, the fluorescence of D-centers is excited andfluorescence of As is measured. Therefore, in this embodiment, only thefluorescence occurring due to FRET is studied. The movement of extendedobjects is arranged so that all portions of each object followapproximately the same path. Since every object includes more than oneunit-specific marker (A or D), each fluorescence emission arising frominteraction of the first fluorophore of every object with a station isaccompanied by the emission arising from interaction of the secondfluorophore of every object with the station. Because all of the objectsare identically (or similarly, as discussed below) labeled, the A or Dgroups are separated by the same distance. If the velocities of all ofthe extended objects are approximately the same, then the correspondingtime intervals ΔT_(i) of different molecules will also be approximatelythe same, meaning that FRET events are correlated for the group ofobjects being analyzed. Therefore, the time ΔT can be determined bycalculating the autocorrelation function G(t) of the measuredfluorescence time dependence, which will have a peak at t=<ΔT>, where<ΔT> is an average value of all corresponding ΔT_(i) measured. The widthof the peak at t=<ΔT> provides the width of the distribution of ΔT forthe measured system.

The temporal resolution δt of the measurement of the I(t) dependenceshould be ≦0.1ΔT in order to obtain information about the distributionwidth of ΔT (variations in ΔT values for different molecules moving inthe studied volume) in a real system. For very short DNA fragments of 10base pairs (3.4 nm in length), the value of ΔT is on the order of 10⁻⁴ sfor DNA moving at a velocity of V=10⁴ nm/s, which is typical of standardelectrophoresis conditions. For longer DNA fragments, the time will beproportionally longer. Hence, a time resolution of 1-10 microseconds issufficient to analyze DNA.

The total time of measurement T depends upon the total number ofcorrelated events that must be measured in order to obtain the desiredS/N ratio. If the distribution width of ΔT is narrower than the timewindow of measurement, then N_(corr)≧100 correlated events arepreferably measured so that S/N≧10. If the distribution width of ΔT iswider than the time window of measurement, more events should bemeasured to have the same S/N ratio at the maximum of G(τ).

It is not necessary to calculate the autocorrelation function G(τ) forthe whole range δt<t<T. The range δt<t<T_(eff)>>10ΔT_(max) is sufficientto determine the time intervals ΔT≦ΔT_(max) where ΔT_(max) is themaximal interval for a given distribution of labels on the DNA sample.The expected resolution of the proposed systems (see below) is δL=10 nm,which corresponds to the length of a DNA helix of 30 base pairs.Therefore, a dynamic range of 10³ (which means 10³ΔT_(min)≧ΔT_(max)) issufficient to analyze DNA fragments of 10⁴ base pairs or shorter. Theconditions δt≦0.1ΔT_(min) and T_(eff)=10ΔT_(max) give minimal dynamicrange 10⁵ for the I(t) measurement sufficient for the study of DNAfragments of 10⁴ base pairs or shorter at a given resolution.Integrators capable of measurement of 64,000 points per I(t) curve arecommercially available. In one embodiment, a better S/N ratio, isobtained from many separately measured I(t) dependences (for the samesystem under the same conditions) of the minimal length T_(eff) that arecombined.

The peak width Δτ on the G(τ) at τ=ΔT is determined by variation of theΔT values for different molecules moving in a real system. The width Δτ,which can be estimated from the width of a band on a polyacrylamide gelcorresponding to a particular DNA fragment (see below), limits themaximal size of the DNA fragment that can be analyzed with thisapproach.

For example, if the first and last units of a 6-unit polymer arelabeled, then a series of bursts of emitted photons will be detectedarising from the polymers wherein the coupled bursts have a constantseparation interval ΔT resulting from the photons emitted by the firstand last labeled units on each polymer. Using the autocorrelationfunction to analyze this data set, the data are resolved from themultiple polymers to identify that there is a 6-unit spread between thefirst and second labels on the polymer. If it is known that the firstand second labels on the polymer correspond to the first and last unitsof the polymer, then the autocorrelation analysis indicates that thepolymer is 6 units long.

In one embodiment, the method of the invention is used to determine thevelocities of penetration of objects, e.g., drugs, toxins, biopolymers,etc., through biological membranes or through protein channels. Forexample, the microscopic speed of transmembrane transport through aprotein pore is determined by the method of the invention. Where afloating fluorophore is a donor and a pore-forming protein is labeledwith acceptors, the situation is mathematically equivalent to thatpresented in FIGS. 1A-1C. Indeed, the two acceptor labels at either endof the pore are permanently separated by distance L and their velocityrelative to the D-center is V, where V is the velocity of the floatingfluorophore through the channel of the protein, and the floatingD-center is always within the Förster distance of the channel walls. Thedistance L is often known from independent structural analysis (Gouaux(1997) Curr. Opin. Struct. Biol. 7:566-573) and so V is easilydetermined.

In another embodiment, the determined time interval ΔT separating thecorrelated events is used to calculate the distance between the labelswhen the velocity of the object is known, or vice versa. The velocity ofan object as it moves past a station is easily determined. For instance,in one embodiment where the object is a DNA molecule, the velocity of acontrol DNA molecule of known length and sequence are measured. The endsof the control DNA molecule are labeled and the methods of invention areperformed on the control molecule. Since the distance between labels onthe control molecule is the length of the molecule, which is known inthis embodiment, the autocorrelation function analysis determines thevelocity of the DNA molecule based on the amount of time required forthe two FRET events to occur that correspond to the ends of the moleculepassing the station. Once the velocity of the control DNA molecule hasbeen measured under a particular set of experimental conditions, themethods of the invention are subsequently used to determine the distancebetween two or more labels on an unknown DNA analyzed under the samegeneral experimental conditions. Once the velocity of the unknown DNAmolecule is known, the distance between two or more labels on theunknown molecule is determined.

In another embodiment, both the rate of movement of an experimental DNAmolecule and the distance between two or more units or unit-specificmarkers in the same molecule are determined by using sets of acceptorsthat emit light at different wavelengths. The end units of the DNAmolecule are labeled with one set of acceptors, which emit light at afirst wavelength, and two or more internal units are labeled with asecond set of acceptors, which emit light at a second wavelength. Inthis embodiment, the time dependence I(t) is measured for thefluorescence at the first wavelength of the labeled end units andprovides information on the velocity of the molecule. The rate is thenused to calculate the distance between the labeled interval units basedon the time dependence I(t) measured from the fluorescence at the secondwavelength.

In yet another embodiment, a mixture of two samples is runsimultaneously. In the first sample, the DNA is labeled at the ends withone set of labels that emit light at a first wavelength. In the secondsample, the same DNA molecule is labeled at two or more internal unitswith a second set of labels that emit light at a second wavelength. Thisexperimental arrangement provides substantially the same set of signalsdescribed above.

The velocities of all extended objects being measured and used tocalculate a particular autocorrelation function are preferablyapproximately the same. The distribution of velocities affects thesensitivity, accuracy, and resolution of the results determined by themethod of the present invention. A more narrow distribution ofvelocities results in a more narrow peak in G(τ) at τ=<ΔT>. Providedthat the same proportion of correlated events is detected, theintegrated area of the peak should be the same for wider and narrowerpeaks. Therefore, a sharper peak in the autocorrelation functioncorresponding to a narrower velocity distribution ensures a bettersignal/noise (S/N) ratio, i.e., the signal is more discernable above thebackground noise. The widths of peaks in the autocorrelation functionalso determine the minimal time intervals between labels on the objectthat are detectable. Under typical slab electrophoretic conditions, thevelocity of short (≦1000 base pairs) DNA fragments is 10⁻⁴-10⁻⁵ m/swithin the gel matrix, which is used below in further calculations.

An estimate of the velocity distribution within a gel matrix can beobtained from the maximal resolution of the gel separation of DNAfragments according to the following analysis. In electrophoresis ofrestriction fragments in slab gels, fragments of lengths 300 and 301 canbe resolved. Within reasonable approximation, the length differences ofDNA fragments is proportional to their velocity differences in gels. Thedistribution of velocities is not wider than the width ofelectrophoretic bands of the resolved fragments. Therefore, the width ofthe velocity distribution inside a polyacrylamide gel matrix is lessthan 0.3%. This is an overestimate, since other things, such asdiffusion of the DNA fragments during the time of electrophoresis,contribute to the widening of bands in a gel. Moreover, the velocitiesestimated from electrophoresis mobility are actually macroscopicallyaveraged velocities and the distributions are also widened byinhomogeneity of the gel matrix. A more narrow distribution of themicroscopic velocities is expected. Indeed, the resolution of fragmentsof up to 1,000-2,000 bases that differ in length by a single base isachieved in capillary electrophoresis (Sinclair (1999) The Scientist 15(9): 18-20), which narrows the velocity distribution down to below 0.1%.

As described in greater detail below, any polymer may be analyzed withthe methods of the invention. In a preferred embodiment, the objects arerod-like extended molecular complexes. In general, a simple covalentchain-like structure of a polymer is too flexible to ensure rigidityover any significant length. Non-covalent interactions (hydrogen bonds,hydrophobic attraction, dispersion forces, etc.) are needed to form andmaintain very extended, stiff, rod like molecular structures. Examplesof such structures are plentiful in biology and include, but are notlimited to: double stranded DNA, muscle proteins (tropomyosin, myosin),proteins of skin and bones (collagen), viral proteins of the infectionsystem (hemagglutinin of grippe virus), and poly-glutamic acid under theconditions promoting formation of long a-helices. The spatial anisotropyof rod-like objects ensures their self-alignment along a flow directionwhen they are put in motion within liquid media. Indeed, any deviationfrom this direction results in a pressure component that restores theoriginal alignment due to solvent flow. Thus, the methods of theinvention can be performed in virtually any liquid if the extendedobject has a sufficiently high velocity. The velocity limitation isdetermined by the following condition: a fluctuation of the labeldistance from the station should not exceed the characteristic scale,which is dependent on the type of object-dependent impulse analyzed.,e.g., the Förster's radius in the case of FRET. While rigidity of anextended object is preferable, it is not an essential property, and themethod of the invention may be used with non-rigid objects. The distanceconstraints can be enforced by using various sample devices (see Section5.3 below and FIG. 2).

In order to employ FRET in the methods of the invention, both the unitspecific marker and the station include fluorophores. In one embodiment,they are single fluorophores. In another embodiment, either the marker,or the station, or both include a plurality of fluorophores. Indifferent embodiments, fluorophores can be fluorescent organic dyes,ions of lanthanide elements, or nanocrystals (or nanoparticles, orquantum dots).

Numerous organic dyes are known in the art. Many of them are availablewith special reactive groups to form the conjugates of the dyes withdifferent objects (see for example Haugland (1996) “Handbook ofFluorescent Probes and Research Chemicals”, Molecular Probes, Inc.). Theconjugates can be formed through covalent and non-covalent linkage.Examples of organic fluorescent dyes that can be used to label extendedobjects or stations include xanthene dyes, BODIPY™ dyes, coumarin dyes,rhodamine dyes, and fluorescein dyes. Other fluorescent compounds arewell-known to those skilled in the art and can be found, e.g., inHaugland (1996) Handbook of Fluorescent Probes and Research Chemicals,Sixth Edition, (Molecular Probes, Inc.).

Lanthanide ions provide several advantages for FRET measurement. First,the energy transfer distance is larger than for most organic dye pairs(Z_(F)=7 nm for lanthanides in D₂O in contrast to Z_(F)=2-5 nm fororganic dye molecules). Second, the excited states of lanthanides havelong lifetimes (0.1-1 ms), which allows for ease of measurement intime-gated fluorescence experiments. One disadvantage of lanthanides isthat their fluorescence is quenched in water by radiationless energytransfer between the excited lanthanide ion and the water molecules inthe coordination sphere. In order to overcome this problem, lanthanidesfor fluorescent applications are coordinated to bulky, hydrophobicligands in order to isolate them from direct contact with water (Saha etal. (1993) J. Amer. Chem. Soc. 115:11032-11033). A second disadvantageof using lanthanides as fluorophores is their relatively low extinctioncoefficient. In order overcome this problem and increase the absorptioncross-section of lanthanide ions, they are coordinated to ligands thathave strong absorption (Selvin & Hearst (1994) Proc. Natl. Acad. Sci.U.S.A. 91:10024-10028; Selvin et al. (1994) J. Am. Chem. Soc.116:6029-6030).

Nanocrystals are tiny pieces of inorganic semiconductor crystals (e.g.,CdSe or InAs) with sizes ranging from single nanometers up to hundredsof nanometers (Alivisatos (1996) J. Phys. Chem. 100: 13226-13239).Depending upon their size and material, nanocrystals emit in differentregions of the electromagnetic spectrum, even when excited with the samewavelength (Bruchez et al. (1998) Science 281: 2013-2016). Specialcoating procedures are applied to stabilize them in solution and makepossible their conjugation with different objects (Chan & Nie (1998)Science 281: 2016-2018). The advantage of nanocrystals is their highbrightness of emission and high stability against photobleaching. Onedisadvantage of the nanocrystals is their relatively large size incomparison with organic dyes and chelated ions, which decreasesresolution. Another disadvantage of nanocrystals is that the stabilizingcoating increases the donor-acceptor separation, which may decrease FRETefficiency.

Donor-acceptor fluorophore pairs are preferably chosen such that theemission spectrum of the donor overlaps with the excitation spectrum ofthe acceptor (Selvin (1995) Meth. Enzymol. 246: 300-334; Wu & Brand(1994) Anal. Biochem. 218: 1-13). Examples of such preferreddonor-acceptor fluorescent dye pairs include, but are not limited to:fluorescein and Texas Red®; Oregon Green™ and Texas Red®; fluorescein(or Oregon Green™) and x-rhodamine; and tetramethylrhodamine and TexasRed®. More preferably, the donor-acceptor dye pairs include, but are notlimited to: Alexa™ 488 and Texas Red®; Alexa™ 488 and x-rhodamine;tetramethylrhodamine and Cy5; terbium (Tb³⁺) ion andtetramethylrhodamine (TMR); or europium (Eu³⁺) ion and Cy5.

In some embodiments, the present invention involves the labeling ofproteins. For example, protein channels or molecular motors can be usedin devices at stations in order to guide extended objects past astation. Furthermore, an extended object for analysis by the methods ofthe present invention may be comprised of amino acids. In order to makefluorescent labeling of proteins possible, amino acids with reactiveside chains can be introduced at strategic points by means of, e.g.,protein engineering (Buckle et al. (1996) Biochemistry 35: 4298-4305; dePrat-Gay, (1996) Protein Engineering 9: 843-847). These reactive sidechains can be further fluorescently labeled by techniques known in theart (see, for example, Haugland, “Handbook of Fluorescent Probes andResearch Chemicals” (1996) Chapters 1-3, 7, 15, 18).

Most preferably, the extended object for analysis using the methods ofthe present invention is DNA. Unit-specific markers may be incorporatedinto the DNA molecule to be analyzed in many ways. In one embodiment,fluorescent nucleotide analogs can be introduced into a polynucleotideduring synthesis (Jameson & Eccleston (1997) Meth. Enzymol. 278:363-390). In another embodiment, fluorescent dyes can be attached viareactive groups on the DNA and the dye (Langer et al. (1981) Proc. Natl.Acad. Sci. U.S.A. 78: 6633-6637; Waggoner (1995) Meth. Enzymol. 246:362-373). In yet another embodiment, lanthanide ions can be attached toDNA via chelating groups (Selvin & Hearst (1994) Proc. Natl. Acad. Sci.U.S.A. 91: 10024-10028; Selvin et al. (1994) J. Amer. Chem. Soc. 116:6029-6030; Kwiatkowski et al. (1994) Nucl. Acids Res. 22: 2604-2611;U.S. Pat. No. 5,591,578). Fluorophores can be attached to the ends of aDNA fragment or to particular reactive groups on the nucleobases. Theymay be attached to DNA via natural or artificially introduced reactivegroups, either prior to or after DNA synthesis.

Unit-specific markers may also be short oligonucleotide probes that arecomplementary to a sequence within the DNA molecule to be analyzed.Labeled oligonucleotide probes can be made using any of the techniquesdescribed above. Preferably, the oligonucleotide probe is labeled withany relatively small fluorophore at either the 3′ or 5′ end. In apreferred embodiment, oligonucleotide probes hybridize to target nucleicacids to form duplexes, triplexes or higher order structures and thelabel is introduced by virtue of the hybridization. Sequence specifichybridization can be performed by art recognized methods (Young andAnderson (1985) in “Nucleic Acid Hybridisation: A Practical Approach”,47-71). An oligonucleotide probe comprised of 15-20 nucleotides willbind specifically to a complementary sequence without random,non-specific binding.

Most preferably, the probes are peptide nucleic acids (PNA), in whichthe phosphate sugar backbone of nucleic acids is replaced by apeptide-like backbone based on the monomer 2-aminoethyleneglycin havingany sequence of nucleobases. This polymer, in contrast to RNA or DNA, iselectrically neutral. A PNA probe can be charged by adding, e.g., afluorescent label, lysine or other positively charged amino acidresidues at one of the termini. An RNA or DNA hybrid with a PNA is morestable than a complex of nucleic acid hybridized with another nucleicacid. Below the melting point, a PNA antisense sequence of ˜20nucleotides binds to its target RNA or DNA within a suitableequilibration time and remains bound to the target sequence for days,even if excess unbound primer is removed.

Both protein and nucleic acid labeling procedures can be used to label aPNA. Furthermore, PNA chimeras with special polypeptide sequences canacquire extra functions. For example, a (His)₆-PNA chimera exhibitsstrong binding to chelated Ni²⁺ ions without compromising its native PNAhybridization properties (Orum et al., (1995) BioTechniques 19:472-480). Potentially, this chelating functionality could be used tolabel the PNA with a lanthanide ion fluorophore.

Oligonucleotide or PNA probes can be used in the methods of the presentinvention for the analysis of DNA sequence. If a labeled probe binds toa DNA analyzed by the methods of the present invention at a specificsite, then the location of the site and its sequence are known. If theDNA molecule itself is not labeled, then only DNA molecules to which atleast two labeled probes are bound will give rise to a correspondingmaximum in the autocorrelation function. The same DNA fragment can befurther analyzed with different probes and the complete sequence of theDNA fragment can be elucidated. This approach is similar to sequencingby hybridization, in which a target nucleic acid sequence is determinedusing data about its hybridization with a specific set ofoligonucleotides (Strezoska et al. (1991) Proc. Natl. Acad. Sci. U.S.A.88: 10089-10093; Bains (1992) BioTechnology 10: 757-758). However, thereis a major difference between this method and methods of the presentinvention. In “classical” sequencing by hybridization, the binding ofprobes to the target DNA is first determined. When all probes capable ofbinding to the DNA are identified, the whole DNA sequence is restored byalignment of the probe sequences, which is done using overlappingfragments of the probes. This is a laborious procedure requiringenormous computer resources (Bains (1992) BioTechnology 10:757-758).This limits maximal length of the target DNA that can be sequenced torelatively short fragments below 1000 bases. In the methods of thepresent invention, not only is information about hybridization of testprobes to a target DNA available, but also the distance between theprobes on the target DNA.

The approach to sequencing is illustrated in FIG. 3A. In a first run,the hybridization of the probes a and b are detected and the distancebetween them is determined. In a second run, the hybridization of theprobes g and d are detected and the distance between them is determined.If a and g are overlapping probes, the relative positions of all fourcomplementary sequences a-d are determined in the target DNA. In oneembodiment, only one test probe is used and the second fluorophore isattached to the terminus of the target DNA (FIG. 3B). In this case, theposition of the tested sequence is determined relative to the labeledend of the target DNA molecule.

For short oligonucleotides, there is a possibility that it willhybridize to more than one complementary site on the target DNA. One wayto decrease the probability of multiple binding sites for a single probeis to use longer probes. Conversely, if the number of complementarysites on the target DNA is small and all peaks in the autocorrelationfunction are resolved, then multiple probe binding sites do not cause aproblem. In this case, all separation distances between the probebinding sites can be determined. The peaks in the autocorrelationfunction correspond to the interlabel intervals and their combinations.If for instance, three probes a, b, and g are bound to the target, thepeaks corresponding to the distances L_(ab), L_(bg), L_(ag),(L_(ab)+L_(bg)), (L_(ab)+L_(ag)), and (L_(bg)+L_(ag)) would occur in theautocorrelation function.

Multiple probe binding can be used for identification properties. Inthis case, shorter probes or a “cocktail” of different probes (see U.S.Pat. No. 5,807,677) can be used. The composition is chosen to ensuremultiple binding. The autocorrelation function will exhibit a complexpattern of peaks whose positions and intensities depend upon the numberof bound copies of each oligonucleotide and the distances between them.To enhance identification ability, different oligonucleotides may belabeled with different fluorophores and several autocorrelationfunctions, each of them measured in the corresponding spectral range,can be used together. It may not be necessary to determine the exactbinding patterns. Rather, the complex pattern of labels can be used as a“fingerprint”, which may be useful in, e.g., forensic analysis.

An advantage of this embodiment of the invention is that the same DNAsample can be used for multiple characterizations. After a run with oneset of oligonucleotide probes, they can be removed from the DNA bytechniques known in the art, e.g., denaturation of the DNA sample underappropriate conditions (see, e.g., Young and Anderson (1985) in “NucleicAcid Hybridisation: A Practical Approach”, 47-71). The dissociatedprobes are removed, e.g., by electrophoresis in the directionperpendicular to that of target DNA movement during analysis, and theDNA is subsequently coupled with another set of probes and is re-run onthe same network of stations, e.g., by reversing the direction ofmovement. The methods of the invention are amenable to this type ofanalysis for two reasons. First, there is no need to concentrate the DNAto form the band; rather a signal volume of any size can be analyzed andis limited only by background intensity. Second, the method isinsensitive to unbound probe, since only oligomers bound to the sameobject can produce a correlated signal.

It is also possible to reanneal the same set of probes to the target DNAand reanalyze it. Thus, the same DNA sample can be relabeled in order toremove photobleached fluorophores. This restored sample can also be runin another region of the matrix where the stations have not yet beenused and therefore are not photobleached. The opportunity for multipleuse of the same sample in the methods of the invention either to enhancestatistics or for complementary analyses allows the use of small amountsof sample (potentially down to the single molecule level) for elaborateanalyses.

The methods of the invention can also be used for multiplex detection ofindividual gene targets and determination of how far they are separatedon the same DNA molecule. Both single- and multiple-probe approaches arepossible. When applied to several different target nucleic acids, themethods of the invention permit the detection of multiple gene targetsin the same test sample. In one embodiment, degenerate or partiallydegenerate probes can be used. This multiplex detection allowsdetermination of the degree of genetic identity in geneticallyuncharacterized organisms. In another embodiment, degenerate probes canbe designed to hybridize to the 3′ and 5′ ends of repeated sequences soas to detect an undetermined number of the repeated sequence, manycopies of which are dispersed throughout the genome. A complex patternin the autocorrelation function results from the analysis and may serveas a “fingerprint” of the studied DNA. The same fluorophore can be usedto label different oligonucleotide probes, and the fingerprint is thedistribution of distances between recognized sites. In a preferredembodiment, several sets of oligonucleotide probes labeled withdifferent fluorophores are used.

The methods of the invention can also be used to determine if twofragments have been joined during a ligation reaction. For this, twooligonucleotide probes, each specific for a sequence on one of twofragments to be ligated, are used. The same fluorophore is used to labelboth oligonucleotide probes. When ligation of the fragments bound to theprobes occurs, a peak appears in the autocorrelation function, since theprobes are now labeling one extended object.

5.3 Apparatus for Correlated Fred Analysis

FIG. 4 depicts a schematic diagram of an optical setup for themeasurement of correlated FRET. A laser is used as an excitation lightsource, LS. Various ion lasers can be used, including but not limited toargon lasers (major lines at 488, 514.5, 351.1, and 363.8 nm), kryptonlasers (major lines at 568.2, 647.1, and 752.5 nm), copper lasers (majorlines at 510 and 578 nm), helium-neon lasers (major lines at 543.5,594.1, 611.9, and 632.8 nm), and helium-cadmium lasers (major lines at325 and 441.6 nm). In different embodiments, either one of the majorlines or (in some cases) a combination of the neighboring lines can beused. In other embodiments, crystal and diode lasers can used in theirmultiple modes, which include but are not limited to nitride blue diodelasers such as InGaN or AlGaInN (emission between 390 and 425 nm),Nd:YAG lasers (266(4×), 354.7(3×), 532(2×), and 1064 nm), Ti:sapphirelasers (330-600(2×) and 660-1200 nm), and alexandrite lasers(360-400(2×) and 720-800 nm). In another embodiment, pulsed lasers withhigh repetition rates (³10 kHz are preferred, ³1 MHz are most preferred)are used. In yet another embodiment, powerful arc lamps, e.g.,high-pressure xenon, mercury, or mercury/xenon, are used. Mostpreferably, a continuous wave (CW) laser is used as the excitation lightsource.

The excitation light, e.g, laser beam, passes through attenuator AT andline filter LF to modulate the light intensity and select a properexcitation line, respectively. After passing through the beam expanderE, the excitation light is reflected from dichroic mirror DM toward thesample mounting. The dichroic mirror reflects light of the excitationwavelength and transmits light of the acceptor emission wavelength. Theexcitation light is focused on the sample S by means of the sample lensSL. In one embodiment, the sample lens is an aspheric lens. In anotherembodiment, it is a microscope objective. The sample comprises anassembly of labeled extended objects moving through a network ofD-centers (see Section 5.3 for the articles of manufacture that arrangesamples for correlated FRET analysis). As sample emission occurs in thefocus of the sample lens SL, it is collected by the sample lens andexits it on the opposite side as a parallel beam. The emission light isdirected through the dichroic mirror, bandpass filter BF, and collectingdetector lens DL. The detector lens focuses the emitted light on thepinhole P that performs as a spatial filter (confocal opticsarrangement), cutting out a large portion of background photons. Thefiltered emitted light is directed to a photodetector PD. Those who areexperienced in the art can appreciate introduction of a notch-filter (tocut the excitation light) and shutters in front of the light sourceand/or photodetector for further filtering (neither notch-filter norshutters are shown in FIG. 4).

Simple optical elements like attenuator, beam expander, pinholes, simplelenses, sample compartment and other light protected components areavailable from many manufacturers (Newport, Oriel Instruments, RolynOptics, Thorlabs Inc., to enumerate few). Optical filters and dichroicmirrors for various spectral ranges can be ordered from ChromaTechnology Corp, Omega Optical Inc., and Kaiser Optical Systems Inc.Microscope objectives can be ordered from Carl Zeiss, Inc. or fromNikon, Inc. It is important to use components with minimal backgroundfluorescence. All lenses, filters, attenuators and mirrors aremanufactured from fused silica (quartz) in the preferred embodiment. Ina preferred embodiment, low-fluorescent, infinity corrected, specializedmicroscope objectives, such as Plan-NEOFLUOAR 40×/NA 1.30 (oilimmersion) or FLUAR 40×/NA 1.30 (oil immersion), from Carl Zeiss, Inc.can be used. For deep blue and ultraviolet excitation light, aUV-transparent microscope objective with water immersion, such asC-APOCHROMAT 40×/NA 1.2, is preferred.

The pinhole diameter depends upon the parameters of the other opticalcomponents used. In one embodiment (FIG. 4), the laser beam diameter is0.7 mm, a 3× beam expander is used, the sample lens is aspheric with aneffective focal length of 14.5 mm, and the detector lens is plano-convexwith a focal length of 100 mm. In this configuration, the diameter ofthe illuminated active spot on the sample is £0.05 mm. Hence, thediameter of the pinhole should be ³0.35 mm. In another embodiment, thePlan-NEOFLUOAR 40× microscope objective is used instead of an asphericlens. In this case, the size of the illuminated spot on the sample is1.2 μm. Hence, the diameter of the pinhole should exceed 30 μm. In apreferred embodiment, pinholes are used that have diameters that areabout 1.5 times larger than the diameter of the image of the illuminatedactive spot on the sample to provide an optimal combination ofrestriction of background and intense sample emission. In this case,pinholes with diameters of 0.53 and 0.045 mm should be used with theaspberic lens and microscope objective, respectively. Either theaspheric lens or the microscope objective should be used as the samplelens to provide maximal collection efficiency of sample emissions.

The photodetector used in the optical setup should be sensitive enoughto detect single photons, it should have high quantum efficiency in thespectral region where the acceptor fluorophore emits and low backgroundnoise. In a preferred embodiment, a photomultiplier tube (PMT) is used.In a more preferred embodiment, the PMT should have an AsGa photocathodefor high quantum efficiency in a wide spectral range. For example, PMTR943-02 (Hamamatsu) has a quantum efficiency greater than 15% atwavelengths of less than or equal to 570 nm and a quantum efficiencygreater than 10% at wavelengths of less than or equal to 840 nm. Atypical PMT of this type has dark current of less than 5 cps (counts persecond) with its photocathode cooled below −30° C. To process the signaloutput, the PMT in photon counting mode is better used with a wide bandpreamplifier (e.g., SR445 of Stanford Research Systems) anddiscriminator. The discriminator is often included in the inputcircuitry of some multichannel analyzers (SR430 of Stanford ResearchSystems) or may by purchased separately (Canberra's Model 2126).

In another embodiment, the detector is an avalanche photo diode (APD).This device is available as part of an integrated single photon countingmodule (SPCM-AQR-15-FC, EG&G Optoelectronics), which includes an APDcooler as well as all electronic circuitry needed for signalconditioning. A typical SPCM module has a quantum efficiency of greaterthan 80% at 600-700 nm and a dark current below 50 cps.

In yet another embodiment, the detector is a multichannel plate PMT(MCP-PMT). This device is related to the PMT and has similar parameters,except for the intrinsic time. While typical intrinsic times of PMTs arewithin the range of 1-10 ns, the intrinsic times of MCP-PMTs can be aslow as 0.1 ns. More preferably, a gateable MCP-PMT (like R2024U,Hamamatsu) is the detector.

In one embodiment, the signal is monitored using CW laser sampleexcitation and continuous detection of the emission signal. In anotherembodiment, a pulse laser can be used for sample excitation. In thisembodiment, detection of emission signal is laser pulse-coupled and thepreferred detection mode is the time-correlated single photon counting(“TCSPC”) mode, which further reduces background due to scattering(Shera et al. (1990) Chem Phys. Lett. 174: 553-557). The laser pulserate depends upon, inter alia, the laser intensity, the lifetime of thefluorophores, and photobleaching effects. A typical pulse rate isbetween 10 and 80 MHz. A gateable MCP-PMT detector is used and is gatedbetween the pulses to detect fluorescence emission, which generally hasa longer half-time than the Rayleigh and Raman scattering thatcontribute to background. In a preferred embodiment, FRET is used todetect the object-dependent impulse, which eliminates the need fortime-gated detection because the emission region is far separated fromthe excitation wavelength and generally does not include solventscattering.

An autocorrelation function for data processing can be obtained eitherduring data collection by using specialized hardware, or bypost-collection computer processing of the data. In one embodiment, anautocorrelation function can be calculated using the Professionalversion of the LabVIEW software package (National Instruments). Inanother embodiment, the methods disclosed in U.S. Pat. No. 5,404,320 canbe implemented in either the software or the hardware in order to obtainan autocorrelation function. In the embodiment depicted in FIG. 4, thetime dependence of the emission is collected in a multichannel analyzerMA (SR430, Stanford Research Systems) during the measurements, istransferred to computer C and is processed with a routine for thecalculation of an autocorrelation function.

The invention also relates to computer system for analyzing an extendedobject labeled with at least two unit-specific markers comprising: acentral processing unit; an input device for inputting a plurality ofobject-dependent impulses of an extended object; and output device; amemory; at least one bus connecting the central processing unit, thememory, the input device and the output device, the memory storing acalculating module configured to calculate an autocorrelation functionfor said plurality of object-dependent impulses of said extended objectinput using said input device.

The invention further relates to a computer program product for use inconjunction with a computer, the computer program product comprising acomputer readable storage medium and a computer program mechanismembedded therein, the computer program mechanism comprising acalculating module configured to calculate an autocorrelation functionof a plurality of object-dependent impulses.

The data generated using the autocorrelation function providesinformation about the extended object. In a preferred embodiment, a setof identical objects having identical labels is examined. The dataproduced by these objects and interpreted using an autocorrelationfunction reveal the identity of patterns of unit-specific markers on theextended objects.

5.4 Sample Devices for Correlated Fret Analysis

Optimization of FRET is dependent upon the spatial proximity of theinteracting fluorophores during the energy transfer. According toformula 1, the fluorescence transfer efficiency is inverselyproportional to the sixth power of Z, the distance between donor andacceptor molecules. The distance at which FRET occurs with 50%probability (the Förster's radius Z_(F)) is between 2 and 7 nm for mostpairs of fluorophores (Wu & Brand (1994) Anal. Biochem. 218: 1-13).Therefore, in order for optimal FRET interaction between the moleculesof the dye pair to occur, the label (either D or A) on the polymer mustpass within 2-7 nm of its partner (either A or D) at the station. Thosewith skill in the art will recognize that, when donor molecules are onthe extended objects, acceptor molecules will be at the stations, andalternatively, when acceptor molecules are on the extended objects,donor molecules will be at the interaction stations. Therefore, althoughthe embodiments may be described with donors at the stations andacceptors on the extended object, it will be understood that theseembodiments also encompass experimental set ups with acceptors at thestations and donors on the extended object.

Described in this section and shown in FIGS. 5A-5C are articles ofmanufacture for correlated FRET analysis of extended objects. Althoughall discussions here involve articles of manufacture for correlated FRETanalysis, it will be understood by those with skill in the art thatsimilar configurations with minor changes, e.g., station types ordimensions, can be used for monitoring other physical effects that areobject-dependent impulses.

Several possible devices for accomplishing correlated FRET methods areset forth in more detail below. For example, a lattice with scatteredD-centers, channels with D-centers at their periphery, or a molecularmotor comprising a D-center or in the vicinity of a D-center can be usedto accomplish the methods of the invention. Any device that will promotethe linear passage of all acceptors bound to a single extended objectpast the same D-center in order to produce object-dependent impulses iscontemplated by the invention. When more than one D-center is analyzedaccording to the methods of the invention, the plurality of D-centersshould be distributed so that there is no interference of the signals.The distribution of the D-centers is adjusted so that the time intervalΔT between the events arising as a result of different acceptors on thesame object passing in interactive proximity with the one D-center isshorter than the time interval between the events arising as a result ofone acceptor passing in interactive proximity to different D-centers.

In one embodiment, the extended objects move through the gel thatincludes D-center stations bound covalently to the gel network (FIG.5A). In this case, the function of the gel matrix is to organize andsupport a spatially distinct network of stations. This approach isfeasible only if the tangential displacement (in the directionperpendicular to motion) of the extended objects during DT, the timeinterval between subsequent passes of acceptors attached to a singleextended object past the same D-center, is small enough not to changethe optimal FRET distance dramatically. Although longitudinal movementof the extended object is forced, the tangential displacementfluctuates, and hence, decreases with decreased DT. The interval DT canbe decreased by increasing the longitudinal speed ol the extendedobject, e.g., by increasing the voltage of the electric field used asthe driving force. Increased longitudinal speed will also increasesolvent resistance to movement of the extended object, which in turnfavors a small molecular cross-section in the longitudinal direction andtherefore helps to eliminate tangential movement of the extended object.Increased solvent resistance can also be accomplished by changing, interalia, the solvent viscosity, temperature, gel concentration.

The same system with a higher concentration gel can be used to analyzelonger and more flexible extended objects. Denser gel networks not onlysupport the network of spatially distinct stations, but also direct themovement of the extended object so that the entire object follows thesame pathway past stations through the gel. In this case, the speed ofthe extended object V is used to maintain the spatial separation L_(i)of the labels on the extended object that corresponds to time intervalt_(i) determined from the autocorrelation function. In order todetermine the value of V, different acceptors A₂ are placed on anextended object at sites separated by a known distance. The dependenceI(t) is measured in the spectral range of these A₂ acceptors and can beused to determine the proper value of V for the configuration.

Most preferably, the gel material is polyacrylamide. Efficient mobilityof DNA fragments of up to 12,000 base pairs in length in non-denaturingconditions was reported (Heiger et al. (1990) J. Chromatogr. 516: 33-48)within polyacrylamide matrices. An example of introduction offluorophores into a gel network is presented below in Example 1.

The resolution of FRET analysis is now calculated for a devicecomprising a low concentration gel with stations and for examination ofrelatively rigid extended objects. For a particular configuration, thepotential resolution of FRET analysis is determined by the distancesbetween acceptors and donors within which FRET can occur (FIG. 6). Foreach trajectory of an acceptor, there is the shortest distance to thenearest donor center that corresponds to the maximal efficiency of FRET.Before and after the acceptor reaches this position, the energy transferis less efficient. For the following calculation, the boundary of theFRET region is where the efficiency of energy transfer is ⅕ of themaximal efficiency. Energy transfer to the acceptor beyond this FRETregion is neglected. The size of the FRET region dL determines thegeometric resolution of the method because the energy transfers withinthe FRET region are indistinguishable from one another.

The effective size of the D-center δL_(D) is the sum of the geometricsize of a donor molecule and of the amplitude of its displacementrelative to the acceptor. The displacement is considerable when both Dand A groups are attached via flexible linkages. In the typical case,dL_(D) is 1-5 nm. For the purpose of this calculation, the shortestseparation between the moving acceptor and the D-center is Z₀=5 nm (atypical Förster radius Z_(F) value). The border of the FRET region δL iswhere the distance (Z₀+ΔZ) separates the acceptor and D-center. Thevalue of ΔZ is determined from the condition that FRET is 5 times lessefficient at (Z₀+ΔZ) than at Z₀. Assuming a dipole-dipole interactionmechanism, the efficiency ratio is ((Z₀+ΔZ)/Z₀)⁶=5, which givesΔZ=0.308Z₀. The distance δL_(Z) between the acceptor positions with theseparations of Z₀ and (Z₀+ΔZ) from the D-center is ((Z₀+ΔZ)²−Z₀²)>>(2Z₀ΔZ)=0.784Z₀. The overall size of the FRET regionδL=δL_(D)+2δL_(Z)=9-13 nm. These estimations are done for acceptordisplacement along the direction of the extended object movement. If thetangential displacement of acceptors is large relative to Z_(F), theefficient value of δL_(Z) will be diminished. Such displacementdecreases the proportion of correlated FRET events.

In another embodiment, a spatial network of stations is organized usinga matrix of beads (FIG. 5B). When densely packed, the beads formchannels of defined sizes due to hexagonal close packing. The movementof the extended object through channels between the beads is similar tothe movement of a molecule through a densely packed column matrix, withthe exception that the diameter of the beads used in this embodiment ofthe present invention is much smaller than the diameter of beads used inconventional separation techniques, e.g., high performance liquidchromatography columns. Preparations of highly ordered lattices of beadswith diameters between 180 and 1500 nm are known in the art (Holland etal. (1998) Science 281: 538-540; Wijnhoven & Vos (1998) Science 281:802-804).

The size of the channels between the beads, and therefore the distancebetween stations on the beads and unit-specific markers on the extendedobjects traveling through the channels, is adjusted by choosing beads ofa given diameter. For example, given beads that are ideal spheres of 65nm diameter that pack into an ideal hexagonal lattice, any point of theextended object travelling through a channel in this lattice will alwaysbe located within 5 nm of the surface of the nearest bead. Polystyrenebeads with diameters as small as 20 nm are commercially available.

The stations within the lattice can comprise anything that will producelabel-dependent impulses when the labeled extended object passes withininteractive proximity of the station. Preferably, the stations in thelattice comprise fluorescent units. In a preferred embodiment, thelattice comprises fluorescent beads or beads labeled with fluorescentdonors or acceptors. The small size of the beads and the fact that theirrefractive index matches that of the solvent decrease light scattering,and are therefore advantageous for light penetration into the matrix. Asa result, the excitation radiation can penetrate at least severalmicrons into the packed bead matrix. The movement of the extended objectthrough the bead lattice exactly resembles the movement of the extendedobject through a dense gel network. An estimation of the resolution ofFRET analysis using a bead lattice is calculated in Example 1 below.

Polystyrene beads having a variety of diameters from 20 nm to 1 mm arecommercially available (Bangs Laboratories, Inc., Fishers, Ind.;Molecular Probes, Inc., Eugene, Oreg.). The beads may already havefluorophores incorporated into them (e.g., FluoSpheres of MolecularProbes, Inc.) or covalently conjugated to their surface (BangsLaboratories, Inc.). Alternatively, beads with surface reactive groups(e.g., amino groups) can be ordered or prepared according to thetechniques known in the art, and labeling can be performed usingnumerous commercially available reactive dyes. Labeling of polystyrenebeads can be done by swelling the beads in a solution of organic solvent(the proper solvent or solvent mixture is determined according tosolubility tables and the molecular weight of the polystyrene) and awater-insoluble dye. In one embodiment, a bead has only one fluorophoreattached. In another embodiment, a bead is densely labeled and can betreated as a single fluorescent center of the size of the bead. In apreferred embodiment, each bead includes more than one fluorescentcenter.

The design of the container wherein the beads are packed depends uponthe monitoring optics system and the force chosen to move the extendedobjects through the beads. For example, if pressure is used to move theextended objects, the container must be designed to withstand increasedpressure. The container must be transparent in the excitation andemission spectral regions.

An estimation of the FRET region dL (see above in this section) for thelattice of beads is illustrated in FIG. 7. Within the lattice of beadswith a diameter of 65 nm=2R, the moving DNA fragment is never more thanZ₀=5 nm from the nearest bead surface. A portion of fluorescentlylabeled beads is embedded into the lattice, which serve as D-centers. Inthis case, the distance to the D-center is the distance to the surfaceof a fluorescent bead in the lattice andδL=2((R+Z₀+ΔZ)²−(R+Z₀)²)>>(2(R+Z₀)ΔZ)=22 nm.

In yet another embodiment, extended objects can be threaded throughnarrow channels in a membrane, wherein one surface of the membrane iscovered with stations (FIG. 5C). Current technology allows theproduction of channels with diameters as small as 10 nm. The D-centersthat form the stations can be attached to one of the membrane surfacesby, inter alia, covalent bonding (in a molecular monolayer), by theLangmuir-Blodgett technique (molecular mono- or multi-layer), by spincoating (down to 10 nm), etc. All fluorescence of the bound acceptors islocalized in vicinity of the membrane surface in this configuration. Arepresentative protocol for preparation of nanochannels and estimationof the FRET resolution using this embodiment of the present invention isenumerated in Example 1, below. DNA fragments having as many as 48,000base pairs were efficiently moved by electrophoresis through 10 nmchannels without noticeable destruction. Our study of DNA transportthrough the PCTE membrane showed that DNA molecules can be efficientlytransported through the long (5 μm) channels with diameter as small as10 nm. Even DNA fragments as long as lambda phage DNA (48,000 base pairslong) can be transferred through these channels without considerablefragmentation.

For the configuration of a membrane with channels, the estimation ofFRET resolution is essentially the same as for the configuration withthe gel-bound donors (see above in this section). If the DNA fragment(diameter=2 nm) is centered within a channel of diameter 10 nm, theseparation Z₀ between the moving acceptor and the D-center (the layerwith donor fluorophores) is 5 nm. The size of the D-center dL_(D) isactually the thickness of the layer (1 and 10 nm for molecular monolayerand spin-coating fluorescent layer depositions, respectively). Theprediction gives δL=δL_(D)+2δL_(Z)=9-18 nm.

PCT Publication No. WO 98/35012, which is incorporated herein byreference in its entirety, provides a detailed description of variousarticles of manufacture comprising embedded fluorophores that are usefulfor practicing the methods of the present invention.

5.5 Structures for Stretching Polymers

Most biological polymers do not exist in solution in their fullyextended conformations. Rather, intra-molecular interactions cause themto exist in more condensed, coiled conformations in solution. Forexample, a DNA molecule in solution exists in a coiled conformation witha diameter of 10 μm. Without being bound by any theory, it issubstantially more difficult to analyze polymers in condensedconformations than in their fully extended forms. Therefore, it ispreferable to extend polymers for analysis in the devices describedabove in Section 5.4.

Structures that allow polymers of any length, including nucleic acidscontaining entire genomes, to be stretched into a long, linearconformation for further analysis may be used in conjunction with themethods and apparatuses of the present invention (see U.S. patentapplication Ser. No. 60/149,020 entitled “Methods And Apparatuses ForStretching Polymers,” inventors Rudolf Gilmanshin and Eugene Chan, filedon even date herewith and incorporated herein by reference in itsentirety). Polymers are loaded into a device and run through thestructures, propelled by, inter alia, physical, electrical or chemicalforces. Stretching is achieved by, e.g., applying shear forces as thepolymer passes through the structures, having the polymer trace out atorturous path, placing obstacles in the path of the polymer, orcombinations thereof. Because the forces are applied continuously, it ispossible to stretch out polymers to a length that is equal to or greaterthan the active area of the apparatus, i.e., where information about thepolymer is collected as the polymer is analyzed. Since multiplemolecules may be stretched in succession, extremely high throughputscreening, e.g., screening of more than one molecule per second, isachieved.

The structures comprise two components: a delivery region and a regionof polymer elongation. The delivery region is a wider channel that leadsinto and out of the region of polymer elongation. The region ofelongation comprises at least one of four main components: (1) funnels;(2) structures having branched channels; (3) channels with bends orcurves; and (4) obstacles defining small gaps. The structures maycomprise combinations of the four main components and variations of themain components themselves. A combination of two or more of the maincomponent features can give rise to additional designs that work well toextend and stretch polymers, particularly DNA, in a controllablefashion. In addition, several of the same design may be repeated inparallel or in series.

Examples of structures (FIG. 8) that can be used in conjunction with themethods and apparatuses of the present invention include, but are notlimited to:

i) funnels with a non-linear increase in fluid velocity;

ii) funnels with a linear increase in fluid velocity;

iii) funnels with obstacles defining small gaps as the region of DNAelongation;

iv) funnels with a non-linear increase in flow rate and obstaclesdefining small gaps;

v) funnels with a linear increase in flow rate and obstacles definingsmall gaps;

vi) funnels with mixed obstacle sizes and gaps, including a gradient ofobstacles sizes and gaps;

vii) branched structures having regions of increased flow rates fromconverging channels;

viii) branched structures having multiple regions of increased flowrates from multiple converging channels;

ix) branched structures having obstacles defining small gaps;

x) branched structures which have at least one funnel as one of thebranches;

xi) branched structures with mixed obstacle sizes and gaps, including agradient of obstacle sizes and gaps;

xii) structures which have obstacles which define small gaps and alsobends or curves;

xiii) structures which have obstacles defining small gaps which have aperiodicity (sine patterns, boxcar repeats, zig-zags);

xiv) structures which have obstacles defining small gaps which arenon-quadrilateral polygons;

xv) structures having a mixture of obstacles which define small gaps,e.g., a set of bars defining small gaps juxtaposed to a field of sinepatterns, or a field of triangles, circles, or stars;

xvi) structures having obstacles defining small gaps integrated withfunnels, branched structures, or bends or curves;

xvii) structures having bends or curves in a funnel shape;

xviii) structures having bends or curves with obstacles defining smallgaps;

xix) structures having regions of DNA elongation in series;

xx) structures having regions of DNA elongation in parallel;

xxi) structures having multiple delivery channels with respectiveregions of elongation; and

xxii) structures having three-dimensional geometries involvingembodiments of the other categories; and

xxiii) Structures which are closed loops containing regions of DNAstretching.

The most preferred embodiment is a structure that combines posts withtwo regions of differing funnel designs, as shown in FIG. 9. Pressureflow is the preferred driving force because of the predictable behaviorof fluid bulk flow.

Structures are constructed on a substrate selected for compatibilitywith both the solutions and the conditions to be used in analysis,including but not limited to extremes of salt concentrations, acid orbase concentration, temperature, electric fields, and transparence towavelengths used for optical excitation or emission. The substratematerial may include those associated with the semiconductor industry,such as fused silica, quartz, silicon, or gallium arsenide, or inertpolymers such as polymethylmetacrylate, polydimethylsiloxane,polytetrafluoroethylene, polycarbonate, or polyvinylchloride. Because ofits transmissive properties across a wide range of wavelengths, quartzis a preferred embodiment.

The use of quartz as a substrate with an aqueous solution means that thesurface in contact with the solution has a positive charge. When workingwith charged molecules, especially under electrophoresis, it isdesirable to have a neutral surface. In one embodiment, a coating isapplied to the surface to eliminate the interactions which lead to thecharge. The coating may be obtained commercially (capillary coatings bySupelco, Bellafonte Pa.), or it can be applied by the use of a silanewith a functional group on one end. The silane end will bond effectivelyirreversibly with the glass, and the functional group can react furtherto make the desired coating. For DNA, a silane with polyethyleneoxideeffectively prevents interaction between the polymer and the wallswithout further reaction, and a silane with an acrylamide group canparticipate in a polymerization reaction to create a polyacrylamidecoating which not only does not interact with DNA, but also inhibitselectro-osmotic flow during electrophoresis.

The channels may be constructed on the substrate by any number oftechniques, many derived from the semiconductor industry, depending onthe substrated selected. These techniques include, but are not limitedto, photolithography, reactive ion etching, wet chemical etching,electron beam writing, laser or air ablation, LIGA, and injectionmolding. A variety of these techniques applied to polymer-handling chipshave been discussed in the literature including by Harrison et al.(Analytical Chemistry 1992 (64) 1926-1932), Seiler et al. (AnalyticalChemistry 1993 (65) 1481-1488), Woolley et al. (Proceedings of theNational Academy of Sciences November 1994 (91) 11348-11352), andJacobsen et al. (Analytical Chemistry 1995 (67) 2059-2063).

5.6 Methods for Sample Movement

Mechanical (e.g., pump or plunger), electroosmotic, or electrokineticmeans may be used in the methods of the invention to move the extendedobjects past stations in order to generate an object-dependent impulse.In the case of mechanical and electroosmotic forces, the extended objectis put into motion by the flow of a stream of solvent. In a preferredembodiment, mechanical or electroosmotic forces are used to moveextended objects through a bead lattice or a channel structure, whereextrinsic structural means are provided to direct the object past thestations. Both electrically charged and neutral extended objects may bemoved by these means.

In a more preferred embodiment, electrokinetic forces are used to movecharged extended objects past the stations. In this case, the extendedobject is moved relative to the solvent, which results inself-orientation of extended objects with their long axes parallel tothe direction of movement. Electrokinetic force is especially preferredto move DNA (electrophoresis), which has a negatively charged backbone.It has been shown that rod-shaped polyelectrolytes, and in particularnucleic acids, are polarized in an electric field (Eigen & Schwarz(1962) In “Electrolytes”, Pergamon Press). This results in very largeinduced dipole moments in DNA, which with field strengths of 10³ V/cmleads to complete alignment of the molecule in the field. The relaxationtime for the polarization is about one microsecond. This effect augmentsthe effect of the moving solvent to help orient the DNA.

As a result of all of these effects, the DNA molecule is stronglyoriented and stretched during gel electrophoresis (Bustamante (1991)Annu. Biophys. Biophys Chem. 20: 415-446; Holzwarth et al. (1989)Biopolymers 28: 1043-1058). Under typical electrophoretic conditionswithin a gel matrix in slab electrophoresis, the voltage is 5-50 V/cmand the velocity of short (≦1000 base pairs) DNA fragments is 10⁻⁴-10⁻⁵m/s. When electrophoresis is performed in capillary-like structure wherea small current cross-section facilitates efficient heat dissipation,voltages of up to 200-500 V/cm can be applied and the speed of the DNAmolecule can reach 10⁻³ m/s.

Parameters of polymer movement through the disclosed devices can beoptimized using theoretical models known in the art (Sung & Park (1996)Phys. Rev. Lett. 77: 783-786; Williams et al. (1998) Biophys. J. 75:493-502).

In another preferred embodiment, polymers are moved by establishing apressure head on the side where the polymers enter the sample device,encouraging fluid to flow to the far side of the sample device, openedto atmospheric pressure or maintained at reduced pressure. The pressurehead may come from any device imposing a physical force, such as asyringe pump. In another embodiment of the pressure control system, indevices with a pressure drop of less than atmospheric pressure, one endof the system is pulled with a vacuum, literally sucking material to beanalyzed through the sample device.

In another embodiment, a molecular motor can be used to guide anextended object past a station within interactive proximity of an agentselected from the group consisting of an electromagnetic radiationsource, a quenching source, and a fluorescence excitation source. Amolecular motor is a device that physically interacts with the extendedobject and pulls it past the station. Molecular motors include, but arenot limited to DNA helicases, DNA polymerases, dyenin, myosin, actin,and kinesin. The molecular motor can be in a solution or positioned on asupport. If the molecular motor is positioned on a support, it is notrequired that an agent is attached to the molecular motor. For example,the station may be created by the support itself, for instance, if thesupport is a conductance membrane. Alternatively, the station may be aseparate entity attached to the support such that it is in interactiveproximity with a polymer moving through the molecular motor. Molecularmotors are described in more detail in PCT Publication No. WO 98/35012,in U.S. patent application Ser. No. 60/096,540 entitled “MolecularMotors,” filed Aug. 13, 1998, and in U.S. patent application Ser. No.09/374,414 entitled “Molecular Motors,” inventor Eugene Y. Chan, filedof even date herewith, all of which are incorporated herein by referencein their entirety.

6. EXAMPLES 6.1 Example 1 Preparation of Sample Devices HavingFluorescent Dyes at the Stations

Preparation of High Molecular Weight Polyacrylamide Labeled WithFluorescein

A concentrated solution (20-40 mM) of succinimidyl ester ofcarboxyfluorescein (fluorescein SE) was prepared in DMF(dimethylformamide). A solution of allylamine (H₂C═CH—CH₂—NH₂) wasprepared in 0.1 M sodium bicarbonate (pH=8.3). The concentrated solutionof fluorescein SE in DMF was added to the allylamine solution such thatthe proportion of DMF in the final reaction mixture did not exceed 10%by weight, the final concentration of allylamine was 15 mM, and a 1.5-2molar excess of the allylamine to the fluorescein SE was present in thefinal solution. The reaction proceeded for 1-2 hours in the dark at roomtemperature, and more than 80% of the fluorescent label was attached tothe allylamine via the amino group.

A fluoroscein-labeled polyamide gel concentration was prepared asfollows. After the reaction of fluorescein with allylamine, concentratedsolutions of acrylamide (30% w/w) and electrophoretic buffer (10×TBE)are added directly to the reaction mixture. Actual volumes of the addedsolutions depend on the volume of reaction mixture and are determinedfrom conditions that the final concentration of acrylamide is 5% w/w andfinal dilution of the buffer is 1×TBE. To initiate polymerization, 3 μlof TEMED and 30 μl of 10% ammonium persulfate were added. Polymerizationwas allowed to proceed for 5-7 hours at room temperature. The mixturewas transferred to an Ultrafree-15 Protein Concentrator Unit (Millipore,Bedford, Mass.) with a molecular weight cutoff of 5,000 D. Theconcentrator was spun in a centrifuge at 2,000×g until less than 10% ofthe orginal volume of the gel was left on the membrane. The filtrate,containing non-reacted fluorophore and low molecular weight fractions ofacrylamide, was discarded. The retentate was enriched in high molecularweight fractions of labeled acrylamide. TBE was added to the retentateuntil the original volume was restored. The purification of the labeledgel by centrifugation was repeated 5-6 times. More than 90% of thefluorescein was incorporated into the final labeled gel concentrate,which contained 1.8 mM fluorescein labeled acrylamide.

In order to prepare a polyacrylamide gel for FRET analysis with adesired amount of covalently linked fluorescein stations, a desiredaliquot of the gel concentrate is added to a polymerizing gel mixture.The final concentration of the fluorophore in the polyacrylamide gel islimited by the condition that the average distance between the gel-boundstations should be larger than the maximal separation interval betweenthe labels on the object to be analyzed.

Preparation of a Lattice of Non-Emitting Nanobeads With DistributedFluorescent Beads

A colloidal suspension of monodisperse polystyrene beads is prepared inwater or a desired buffer solution (if the original suspension arrivesat a concentration of <10% it should first be concentrated). Fluorescentbeads are added at this stage and dispersed in the suspension byvortexing. The final concentration of the fluorescent centers within thelattice is limited by the condition that the average distance betweenthe stations should be larger than the maximal separation intervalbetween the labels on the object to be analyzed.

The colloidal suspension is transferred into a capillary tube (a flatcapillary is preferred). A quasicrystallization process is performed bysedimentaion of the beads in a centrifuge at 200-800×g for severalhours. The actual time for lattice growth depends upon the desired sizeof the quasicrystal and upon the diameter of the beads used. It can takeup to several days to grow a quasicrystal that is several millimiterslong.

Preparation of a Membrane With Nano-Channels and a Fluorescent Layer

As a prototype for the nanochannel system for the correlated FRETmeasurements, polycarbonate track-etch membranes (PCTE) is used(Osmonics, Livermore, Calif.). PCTE membranes are microporous screensthat derive their special properties from the manufacturing technology.They are comprised of cylindrical pores of regular diameter that arenormal (within ±35°) to the membrane surface. Pore diameters of 10 nmand larger are available. The distribution of actual diameters of thepores is narrow and varies from +0% to −20% of rated pore size. Typicalpore densities can be as high as 10⁹ pores/cm².

A polycarbonate layer containing 0.4% pyrrometene 580 dye (Exciton,Dayton, Ohio) is cast onto a polycarbonate supporting film by acontrolled-gap blade technique. Fluorescent layers with thickness ofseveral hundreds of nanometers have been prepared in this way. Usingspin coating, fluorescent layers with thickness of tens of nanometersare possible. After the deposition, the film is the sum of two layers,each of them mostly or completely made of polycarbonate. A generaltrack-etch technique is applicable to such membranes (either in house orby order to Osmonics), resulting in a PCTE membrane with nanochannelsand a fluorescent layer.

Alternatively, reactive groups (e.g., amino groups) are introduced ontothe surface of a PCTE membrane with 10 nm channels by techniques knownin the art, or such labeling is performed by order to Xenopore, Inc.(Hawthorne, N.J.). Afterwards, a dye is covalently linked to them, forexample, the succinimidyl ester of Texas Red® can be used with aminogroups.

6.2 Example 2 Calculation of Illumination Intensity Needed to Achieve aHigh FRET Signal to Noise Ratio

The probability of energy transfer between donor and acceptor, P_(tr),is determined by the Förster factor, and is ½ at the Förster distanceZ_(F). The overall number of FRET events, N_(tr), that occur duringT_(tr), the time that donor and acceptor are within interactionproximity, is

N _(tr) =P _(tr) X=P _(tr) X ₀ T _(tr)  (3)

where X is the number of excitations of the donor center during T_(tr),and X₀ is the number of excitations of the donor center per second. IfN_(tr) ³1, the FRET probability is 1 and all FRET events are correlated(i.e. each FRET event has a corresponding correlated FRET event). IfN_(tr)<1, then N_(tr)=P⁰ _(tr), the overall probability of excitationtransfer from a donor to an acceptor at a given illumination intensity.In this case, the probability of a correlated FRET event is P_(corr)=(P⁰_(tr))². If P_(corr)<1, then the excess occurrence of backgroundfluorescence over correlated fluorescence is given by: F=P⁰_(tr)/P_(corr)=1/P⁰ _(tr) per correlated photon.

In addition to these limitations, there are also limitations introducedby the type of apparatus used to measure the FRET. For example, thefinite aperture of the optical system, reflections, and losses onfilters decrease K, the overall proportion of registered photons.Therefore, the probability P_(act) should be used instead of P⁰ _(tr):

P _(act) =KP ⁰ _(tr) =KP _(tr) X ₀ T _(tr)  (4)

To measure N_(corr), the number of correlated photons necessary for ahigh signal to noise ratio, overall I_(tot) of the photons should bemeasured:

I _(tot)=2N _(corr) F=2N _(corr) /P _(act)=2N _(corr)/(KP _(tr) X ₀ T_(tr))  (5)

To achieve a large N_(corr) within a reasonable time of measurement, T,the probability P_(act) should be as close to unity as possible.However, the proportion of registered photons K is always <1 andnon-correlated photons will be always present in excess of 1/K.

Losses in a system having custom made interference filters and adichroic mirror total 40-50% due to reflection and absorption.Additional losses of 10-15% are introduced by the limited width (80 nm)of the bandpass filter in the emission channel. Collection efficiencyfor an aspheric lens (special design, fused silica, NA=0.88) is 0.0525.This results in K=(0.55)(0.85)(0.0525)=0.025=2.5% for this system. If amicroscope objective (Plan-NEOFLUOAR, NA=1.3) is used instead of theaspheric lens, then K=(0.55)(0.85)(0.18)=0.084=8.4%. Furthermore, if PMTR943-02 (Hamamatsu) with GaAs photocathode having a quantum efficiencyof detection >10% at wavelengths >840 nm is used, then the K value isfurther decreased to 0.0025 or 0.0084 for an aspheric lens or microscopeobjective, respectively. For further calculations, the value K=0.05 isused.

For δL=10 nm and a velocity of the extended object of V=10⁴ nm/s,T_(tr)=10⁻³s=1 ms. Assuming that the absorption cross-section of thedonor center is σ=3×10⁻¹⁶ cm², the quantum efficiency of donor centeremission Q=0.5, and at an excitation wavelength of 488 nm (f=2.46×10¹⁵photons/s for 1 mW of power), the density of excitation power, W_(A),needed to provide N_(em) emitted photons within T_(tr) can be estimatedby:

W _(A) =N _(em)/(σQT _(tr) f)  (6)

For N_(em)=100 photons, an excitation density power of W_(A)=270 W/cm²is needed. If the illuminated spot diameter is 0.05 mm (such as thatproduced when using an aspheric lens), then an Ar laser having power ofat least 5.3 mW in the 488 nm line may be used. Faster velocities of theextended object require even more power because T_(tr) decreases. Use ofa microscope objective lens increases the illumination densityconsiderably due to a smaller illuminated spot diameter (microns).However, the maximum possible excitation intensity is limited by thephotobleaching of donor center fluorophores (see, e.g. analysis in U.S.Pat. No. 4,979,824) and optimization of excitation intensity is acomplex process.

It can reasonably be assumed that X₀T_(tr)=10-100 photons corresponds toan optimal level of illumination, which corresponds to a total emissionof 10⁴-10⁵ photons/sec perfluorophore, which results in bleaching withinone second. Further assuming that P_(tr)=0.5 and using equation (5),I_(tot)=8×10⁴ photons should be accumulated to measure N_(corr)=1000photons for optimization of the signal to noise ratio.

6.3 Example 3 The Effects of Background Emission on FRET Measurements

The calculations performed thus far assume that donors and acceptors areideal, that is, they are excited and emit within a finite spectral rangeonly. In actuality, donor fluorophore emission peaks have low intensityshoulders or tails toward the red side of the peaks, while acceptorfluorophore excitation peaks have low intensity shoulders or tailstoward the blue side of the peaks. Therefore, exciting donors will alsodirectly excite acceptors to some extent. Furthermore, in addition toacceptor fluorescence, there is some emission from the donor at the samewavelengths.

Consider the dye pair fluorescein (donor) and Texas Red® (acceptor).Texas Red® emission has maximum at 611 nm. Although the maximum offluorescein fluorescence is at 524 nm, it still has considerableemission (9% of its maximal value) at 611 nm. If the Ar laser line at488 nm is used to excite the fluorescein, it would also directly exciteTexas Red®, although with an efficiency of 2.5% of the maximal value.Another choice of donor can be Alexa™ 488. On the one hand, its emissionspectrum has less overlap with the excitation spectrum of Texas Red®,which makes FRET less efficient than from fluorescein, provided allother conditions are the same. On the other hand, Alexa™ 488 emission at611 nm is only 2% of its maximal value and it is excited moreefficiently than fluorescein at 488 nm. Alexa™ 488 is also much morephotostable than fluorescein. Therefore, the combination of factorsmakes Alexa™ 488 a more preferred donor for the applications herein.

Experiments have been performed on the energy transfer between thefluorescein (donor) and YOYO-3 iodide (acceptor, Molecular Probes, OR).Fluorescein was bound covalently to a polyacrylamide gel (5% gel with0.003% [mol/mol] of fluorescein labeled monomers, see more detailsbelow). Short fragments of DNA (100 to 1000 base pairs) were run throughthe gel in an amount of 50 to 150 nanograms per fraction. The DNA waslabeled with YOYO-3 iodide, which binds to double-stranded DNA (1:10 ofYOYO-3:DNA base pairs) and has a very small fluorescence quantum yieldin its unbound form. Fluorescence was monitored at 641 nm, whereI_(em)(641) is 3.8% of I_(em)max for fluorescein. The I_(ex)(488) ofYOYO-3 is 1.4% of the I_(ex)max. Under these conditions, detectedfluorescein emission at 641 nm was 1.3×10⁶ photons/s while YOYO-3emission was 3×10⁴ photons/s. More than 90% of YOYO-3 emission was dueto FRET. The proportion of the YOYO-3 emission due to direct excitationand due to FRET was calculated by comparison of the emission of YOYO-3excited at 488 and 580 nm in presence and absence of fluorescein,respectively. Fluorescein emission is not excited at 580 nm.

For the tested system, the donor fluorescence was 43 times larger thanthe overall acceptor fluorescence and the fluorescence of the directlyexcited acceptor was only a minor fraction of the FRET-induced acceptoremission. For this experimental configuration and dye pair, resultscould be improved by increasing the concentration of moving acceptorsand decreasing the concentration of donor centers. For an invertedsystem, where the donor is attached to DNA and the acceptor isimmobilized in a spatial network, the opposite concentration ratio wouldbe more advantageous. The actual concentrations of D or A and theirplacement should be chosen for each dye pair according to the spectraland physico-chemical properties of the molecules. The use of Alexa 488in place of fluorescein in this system decreases the donor fluorescenceat 641 nm to 4×10⁵ photons/s.

Other factors contribute to the generation of background signal duringFRET measurements. Among the most important of these are Rayleigh andRaman scattering, which are due to solvent and other materialsconstituting the system. The contribution of Rayleigh and Ramanscattering to background signal is approximately proportional to theilluminated volume used in the experiment. For the smallest volume,several femtoliters, tested for direct dye emission, a S/N ratio of 40was observed. With suppression of the rotational Raman bands of water,the S/N ratio was reported to be as high as 1700 (Eigen & Rigler (1994)Proc. Natl. Acad. Sci. U.S.A. 91: 5740-5747). In the experimentsperformed herein, the scattering contribution to the background is lesssubstantial because the emission is measured at a wavelength that ismore than 100 nm from the excitation wavelength, and background signaldue to scattering is filtered out with donor emission background.

6. REFERENCES CITED

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

What is claimed is:
 1. A method for analyzing extended objectscomprising: (a) moving with respect to at least a first station aplurality of similar extended objects that are each similarly labeled atsimilar positions with at least a first unit-specific marker and asecond unit-specific marker to generate a plurality of object-dependentimpulses as the similar extended objects pass the first station, whereinthe first and second unit-specific markers are at different positionsalong each similar extended object, wherein the similar extended objectsare extended molecules or extended molecular complexes; (b) measuringthe generated plurality of object-dependent impulses as a function oftime, wherein the object-dependent impulses generated due to said firstunit-specific marker of an individual one of said plurality of similarextended objects passing said first station and said object-dependentimpulses generated due to said second unit-specific marker of saidindividual one of said plurality of similar extended objects passingsaid first station are resolved in time; and (c) calculating anautocorrelation function of said measured plurality of object-dependentimpulses, to analyze the extended objects.
 2. The method of claim 1wherein said plurality of similar extended objects are moved withrespect to the first station at similar velocities.
 3. The method ofclaim 1, wherein the plurality of similar extended objects is aplurality of polymers.
 4. The method of claim 3, wherein each polymer inthe plurality of polymers is identical to every other polymer in theplurality of polymers.
 5. The method of claim 4, wherein each polymer inthe plurality of polymers is labeled with identical unit-specificmarkers at identical positions within the polymer.
 6. The method ofclaim 3, 4 or 5, wherein each polymer in the plurality of polymers is anucleic acid.
 7. The method of claim 6, wherein the nucleic acid is DNA.8. The method of claim 1, wherein the autocorrelation function isdefined by the formula: G(τ) = 1/T∫₀^(T)I(t)I(t + τ)  t

where G(τ) is the autocorrelation function of the time dependence ofmeasured object-dependent impulses, T is the total time of measurementof I(t), and I(t) is the object-dependent impulse measurement at eachtime point t.
 9. The method of claim 1, wherein the autocorrelationfunction is defined by the formula:$G_{j} = {\left( {1/N} \right){\sum\limits_{i = 0}^{N}{I_{i}I_{i + j}}}}$

where G_(j) is the autocorrelation function of the time dependence ofmeasured object-dependent impulses at time jΔt, N is the total number ofdata values, I_(i) is the object-dependent impulse measurement at timet_(i), I_(i+j) is the object-dependent impulse measurement at timet_(i)+jΔt, and Δt is a time interval.
 10. The method of claim 1, whereinthe at least a first station is a plurality of stations.
 11. The methodof claim 10, wherein the plurality of similar extended objects is movedthrough a lattice of beads and the plurality of stations is positionedon a subset of the plurality of beads.
 12. The method of claim 8, 9 or11, wherein the plurality of similar extended objects is a plurality ofpolymers.
 13. The method of claim 12, wherein each polymer in theplurality of polymers is a nucleic acid.
 14. The method of claim 13,wherein the nucleic acid is DNA.
 15. The method of claim 10, wherein theplurality of similar extended objects is moved through a channel, saidchannel having a first end, a second end, and at least one wall, theplurality of stations being positioned along the at least one wall. 16.The method of claim 10, wherein the plurality of similar extendedobjects is moved through a channel, said channel having a first end, asecond end, and at least one wall, the plurality of stations beingpositioned at said first end or said second end.
 17. The method of claim15 or 16, wherein the plurality of similar extended objects is aplurality of polymers.
 18. The method of claim 17, wherein each polymerin the plurality of polymers is a nucleic acid.
 19. The method of claim18, wherein the nucleic acid is DNA.
 20. The method of claim 10, whereinthe plurality of similar extended objects is moved simultaneouslythrough a plurality of channels, each channel in said plurality ofchannels having a first end, a second end, and at least one wall, eachchannel having at least one station positioned along the at least onewall.
 21. The method of claim 10, wherein the plurality of similarextended objects is moved simultaneously through a plurality ofchannels, each channel in said plurality of channels having a first end,a second end, and at least one wall, each channel having at least onestation positioned at said first end or said second end.
 22. The methodof claim 20 or 21, wherein the plurality of similiar extended objects isa plurality of polymers.
 23. The method of claim 22, wherein eachpolymer in the plurality of polymers is a nucleic acid.
 24. The methodof claim 23, wherein the nucleic acid is DNA.
 25. The method of claim 1,wherein the plurality of similar extended objects is moved through theaction of at least one molecular motor.
 26. The method of claim 25,wherein the at least one molecular motor is a plurality of molecularmotors in solution.
 27. The method of claim 25 or 26, wherein theplurality of similar extended objects is a plurality of polymers. 28.The method of claim 27, wherein each polymer in the plurality ofpolymers is a nucleic acid.
 29. The method of claim 28, wherein thenucleic acid is DNA.
 30. The method of claim 1, wherein theobject-dependent impulses generated due to said first unit-specificmarker of an individual one of said plurality of similar extendedobjects passing said first station and said object-dependent impulsesgenerated due to said second unit-specific marker of said individual oneof said plurality of similar extended objects passing said first stationare different.
 31. The method of claim 30, wherein the plurality ofsimilar extended objects is a plurality of polymers.
 32. The method ofclaim 31, wherein each polymer in the plurality of polymers is a nucleicacid.
 33. The method of claim 32, wherein the nucleic acid is DNA. 34.The method of claim 1 wherein the object-dependent impulses arefluorescence resonance energy transfer.
 35. The method of claim 34,wherein said first station comprises at least one donor fluorophore andsaid first unit-specific marker and said second unit-specific markereach comprise at least one acceptor fluorophore.
 36. The method of claim34, wherein said first station comprises at least one acceptorfluorophore and said first unit-specific marker and said secondunit-specific marker each comprise at least one donor fluorophore. 37.The method of claim 34, 35 or 36, wherein the plurality of similarextended objects is a plurality of polymers.
 38. The method of claim 37,wherein each polymer in the plurality of polymers is a nucleic acid. 39.The method of claim 38, wherein the nucleic acid is DNA.
 40. The methodof claim 1, wherein repetitive information in the measured plurality ofobject-dependent impulses provides information about the length of theextended objects.
 41. The method of claim 1, wherein analysis of theextended objects provides information about the distance between saidfirst unit-specific marker and said second unit-specific marker on theextended objects.
 42. The method of claim 2, wherein analysis of theextended objects provides information about the velocity of the extendedobjects.
 43. The method of claim 1, wherein analysis of the extendedobjects provides information about the linear arrangement of unitswithin the extended objects.
 44. The method of claim 40, 41, 42 or 43,wherein the plurality of extended objects is a plurality of polymers.45. The method of claim 44, wherein each polymer in the plurality ofpolymers is a nucleic acid.
 46. The method of claim 45, wherein thenucleic acid is DNA.
 47. A method for analyzing extended objectscomprising calculating an autocorrelation function of measuredobject-dependent impulses, wherein said object dependent impulses havebeen produced by: (a) moving with respect to at least a first station aplurality of similar extended objects that are each similarly labeled atsimilar positions with at least a first unit-specific marker and asecond unit-specific marker to generate a plurality of object-dependentimpulses as the similar extended objects pass the first station, whereinthe first and second unit-specific markers are at different positionsalong each similar extended object, wherein the similar extended objectsare extended molecules or extended molecular complexes; and (b)measuring the generated plurality of object-dependent impulses as afunction of time, wherein the object-dependent impulses generated due tosaid first unit-specific marker of an individual one of said pluralityof similar extended objects passing said first station and saidobject-dependent impulses generated due to said second unit-specificmarker of said individual one of said plurality of similar extendedobjects passing said first station are resolved in time.
 48. The methodof claim 47, wherein the extended objects are polymers.
 49. The methodof claim 48 wherein the polymers are nucleic acids.
 50. The method ofclaim 49, wherein the nucleic acids are DNA.
 51. The method of claim 47,wherein the autocorrelation function is defined by the formula:G(τ) = 1/T∫₀^(T)I(t)I(t + τ)  t

where G(τ) is the autocorrelation function of the time dependence ofmeasured object-dependent impulses, T is the total time of measurementof I(t), and I(t) is the object-dependent impulse measurement at eachtime point t.
 52. The method of claim 47, wherein the autocorrelationfunction is defined by the formula:$G_{j} = {\left( {1/N} \right){\sum\limits_{i = 0}^{N}{I_{i}I_{i + j}}}}$

where G_(j) is the autocorrelation function of the time dependence ofmeasured object-dependent impulses at time jΔt, N is the total number ofdata values, I_(i) is the object-dependent impulse measurement at timet_(i), I_(i+j) is the object-dependent impulse measurement at timet_(i)+jΔt, and Δt is a time interval.
 53. The method of claim 47,wherein the object-dependent impulses generated due to said firstunit-specific marker of an individual one of said plurality of similarextended objects passing said first station and said object-dependentimpulses generated due to said second unit-specific marker of saidindividual one of said plurality of similar extended objects passingsaid first station are different.
 54. The method of claim 53, whereinthe extended objects are polymers.
 55. The method of claim 51, 52 or 54,wherein the polymers are nucleic acids.
 56. The method of claim 55,wherein the nucleic acids are DNA.
 57. The method of claim 47 whereinthe object-dependent impulses are fluorescence resonance energytransfer.
 58. The method of claim 57, wherein the extended objects arepolymers.
 59. The method of claim 58, wherein the polymers are nucleicacids.
 60. The method of claim 59, wherein the nucleic acid is DNA. 61.The method of claim 47, wherein the analysis of the extended objectsprovides information about the length of the extended objects.
 62. Themethod of claim 47, wherein analysis of the extended objects providesinformation about the distance between said first unit-specific markerand said second unit-specific marker on the extended objects.
 63. Themethod of claim 47, wherein analysis of the extended objects providesinformation about the velocity of the extended objects.
 64. The methodof claim 47, wherein analysis of the extended objects providesinformation about the linear arrangement of units within the extendedobjects.
 65. The method of claim 61, 62, 63 or 64, wherein the extendedobjects are polymers.
 66. The method of claim 65, wherein the polymersare nucleic acids.
 67. The method of claim 66, wherein the nucleic acidsare DNA.